scholarly journals Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice

Blood ◽  
2011 ◽  
Vol 118 (15) ◽  
pp. 4224-4230 ◽  
Author(s):  
Andrea U. Steinbicker ◽  
Thomas B. Bartnikas ◽  
Lisa K. Lohmeyer ◽  
Patricio Leyton ◽  
Claire Mayeur ◽  
...  
Keyword(s):  
Gene Expression ◽  
Iron Overload ◽  
Hereditary Hemochromatosis ◽  
Bmp Signaling ◽  
Type I ◽  
Anemia Of Chronic Disease ◽  
Hepcidin Expression ◽  
Hepatic Expression ◽  
Bmp Receptors ◽  
Hepcidin Gene

Abstract Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3- than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepcidin expression. Both Alk2 and Alk3 were required for induction of hepcidin gene expression by BMP2 in cultured hepatocytes or by iron challenge in vivo. These observations demonstrate that one type I BMP receptor, Alk3, is critically responsible for basal hepcidin expression, whereas 2 type I BMP receptors, Alk2 and Alk3, are required for regulation of hepcidin gene expression in response to iron and BMP signaling.

Role of BMP Signaling In the Anemia of Chronic Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2043-2043
Author(s):  
Andrea U. Steinbicker ◽  
Ashley J. Vonner ◽  
Chetana Sachidanandan ◽  
Lisa Lohmeyer ◽  
David T. Scadden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Research Funding ◽  
Critical Role ◽  
Bmp Signaling ◽  
Mrna Levels ◽  
Anemia Of Chronic Disease ◽  
Hepcidin Expression ◽  
Hepcidin Gene ◽  
Hepcidin Mrna

Abstract Abstract 2043 Introduction: Anemia of chronic disease (ACD) describes anemia associated with diverse chronic inflammatory, infectious, or neoplastic processes. These conditions are frequently associated with increased circulating levels of inflammatory cytokines such as interleukin 6 (IL-6). IL-6 regulates expression of the hormone hepcidin, which inhibits the release of iron from hepatocytes, macrophages, and enterocytes into the circulation. In addition to IL-6, hepcidin gene expression is known to be transcriptionally regulated by bone morphogenetic protein (BMP) signaling. Hypothesis: We hypothesized that BMP signaling is required for the induction of hepcidin gene expression by IL-6 and plays a critical role in the pathogenesis of ACD. Methods: We used a turpentine-dependent model of ACD in mice. Mice were challenged with weekly subcutaneous injections of turpentine, which induces anemia in an IL-6 dependent manner. This model was studied to determine hepcidin gene expression and rescue ACD using BMP inhibition. Moreover, we examined hepcidin gene expression in zebrafish injected with Pseudomonas aeruginosa, and in transgenic zebrafish overexpressing human IL-6. The regulation of hepcidin gene expression was also studied in the human hepatocarcinoma cell line (HepG2). Results: Injections of mice with IL-6 (0.8 μg/g ip) increased hepatic hepcidin mRNA levels expression at 24 hours and decreased serum iron concentrations. Both effects were prevented by a small molecule BMP type I receptor kinase inhibitor, LDN-193189, or protein BMP antagonists. Weekly turpentine injections induced microcytic anemia after 3 weeks with a decrease in hemoglobin levels from 12.8±0.3 to 9.7±1.7 g/dL (*p<0.01). Concurrent treatment with LDN-193189 prevented turpentine-induced anemia and microcytosis (*p<0.01 for both). In mice challenged with turpentine for 6 weeks, treatment with LDN-193189, beginning after anemia was established at week 3, led to an increase in hemoglobin levels at week 6 (10.9±0.1 vs 9.5±0.2 g/dL, LDN193189 vs vehicle, respectively; *p<0.05). In zebrafish, microinjection with Pseudomonas aeruginosa or overexpression of human IL-6 induced hepatic hepcidin expression, an effect which was blocked by LDN-193189. Incubation of HepG2 cells with IL-6 (100 ng/ml) increased hepcidin mRNA levels 2 to 5 fold. Pretreatment with LDN-193189, or recombinant protein BMP antagonists such as noggin, abrogated the induction of hepcidin expression by IL-6. Incubation of HepG2 cells with BMP6 (2.5 to 10 ng/ml) modestly increased hepcidin mRNA levels. However, the combination of IL-6 and BMP6 synergistically increased hepcidin gene expression (*p<0.05). Conclusion: BMP signaling appears to play a critical role in the pathogenesis of anemia in a mouse ACD model. Our findings support the concept that BMP signaling is required for the induction of hepcidin gene expression by IL-6 in vitro and in vivo. Moreover, manipulation of BMP signaling represents a potentially novel therapeutic approach to the treatment of anemia associated with inflammation. Disclosures: Steinbicker: Deutsche Forschungsgemeinschaft DFG: Research Funding. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Peterson:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Bloch:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Yu:Harvard Stem Cell Institute Seed Grant and the Howard Hughes Medical Institute Early Career Physician-Scientist Award: Honoraria, Research Funding; NHLBI 5K08HL079943: Research Funding.

scholarly journals Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4915-4923 ◽  
Author(s):  
Andrea U. Steinbicker ◽  
Chetana Sachidanandan ◽  
Ashley J. Vonner ◽  
Rushdia Z. Yusuf ◽  
Donna Y. Deng ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Iron Homeostasis ◽  
Iron Bioavailability ◽  
Bmp Signaling ◽  
Neoplastic Disease ◽  
Type I ◽  
Hepcidin Expression ◽  
Hepatic Expression ◽  
Morphogenetic Protein ◽  
Anemia Of Inflammation

Abstract Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6–induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

Alk3, a BMP Type I Receptor Is Required for the Induction of Hepatic Hepcidin Gene Expression by Interleukin-6

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 686-686 ◽  
Author(s):  
Andrea U. Steinbicker ◽  
Claire Mayeur ◽  
Lisa K. Lohmeyer ◽  
Patricio Leyton ◽  
Sonya M. Kao ◽  
...  
Keyword(s):  
Gene Expression ◽  
General Hospital ◽  
Small Molecule ◽  
Iron Metabolism ◽  
Serum Iron ◽  
Small Molecule Inhibitors ◽  
Massachusetts General Hospital ◽  
Bmp Signaling ◽  
Type I ◽  
Hepcidin Gene

Abstract Abstract 686 Introduction: Anemia of chronic disease (ACD), the second most prevalent form of anemia, is commonly associated with chronic inflammatory, infectious, or neoplastic conditions. ACD is characterized by high hepcidin levels that decrease serum iron levels by inducing degradation of the iron exporter ferroportin. In contrast, a relative deficiency of hepcidin leads to ferroportin overexpression and iron overload. Hepcidin is transcriptionally regulated by interleukin-6 (IL-6) and bone morphogenic protein (BMP) signaling. Binding of BMP ligands to type II and type I BMP receptors induces the type II receptor to phosphorylate and activate one of four type I receptors. We sought to identify the type I BMP receptor that participates in the ability of IL-6 to induce hepatic hepcidin gene expression. Methods: The four type I BMP receptors are Alk1, Alk2, Alk3, and Alk6. Alk1 is predominantly expressed in the endothelium. Alk6 is expressed at low levels in murine liver. In contrast, Alk2 and Alk3 are abundantly expressed in hepatocytes. Global deficiency of Alk2 or Alk3 is embryonic lethal. To selectively delete Alk2 or Alk3 in hepatocytes, we studied mice homozygous for Alk2 or Alk3 sequences flanked by loxP sites (Alk2fl/fl and Alk3fl/fl, respectively) that also carried a transgene specifying Cre recombinase under the control of the albumin gene promoter (Alb-Cre). Eight- to 12-week-old male mice (Alk2fl/fl, Alk2fl/fl; Alb-Cre, Alk3fl/fl, Alk3fl/fl; Alb-Cre) on a standard, iron-replete diet were injected via the tail vein with an adenovirus specifying IL-6 (Ad.IL-6) or an adenovirus specifying green fluorescent protein (GFP; Ad.GFP), as a control (1010 particles per ml for both). Seventy-two hours later, mice were euthanized, and blood was obtained for measurement of serum iron levels and transferrin saturations. Livers were harvested, and RNA was extracted. Hepatic levels of mRNAs encoding Alk2, Alk3, hepcidin, heme oxygenase-1 (HO-1, a transcriptional target of IL-6), and Id-1 (a BMP gene target) were measured by qRT-PCR. Hepatic STAT3 phosphorylation (a marker of IL-6 receptor activation) was measured using immunoblot techniques. Results: Liver-specific deletion of Alk2 or Alk3 caused mild and severe iron overload, respectively. Injection of Ad.IL-6, but not Ad.GFP, decreased serum iron levels and transferrin saturations in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. In contrast, infection of Alk3fl/fl; Alb-Cre mice with Ad.IL-6 did not alter serum iron levels and only modestly reduced transferrin saturations. Infection with Ad.IL-6 induced of hepatic hepcidin gene expression in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. Hepatic hepcidin mRNA levels were markedly reduced in Ad.GFP-infected Alk3fl/fl; Alb-Cre mice, and infection with Ad.IL-6 failed to increase hepcidin mRNA levels in this genotype. Ad.IL-6 infection induced hepatic Id-1 mRNA levels in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice, but not in Alk3fl/fl; Alb-Cre mice. Infection with Ad.IL-6 induced hepatic STAT-3 phosphorylation and HO-1 gene expression in all 4 genotypes of mice. Conclusions: Taken together, these results demonstrate that the response to IL-6, as reflected by STAT-3 phosphorylation and induction of HO-1 gene expression, does not require Alk2 or Alk3. In contrast, BMP signaling, predominantly via Alk3, is essential for the induction of hepcidin gene expression by IL-6. These results suggest that selective inhibition of Alk3 may represent a novel therapeutic approach to the treatment of ACD. Disclosures: Peterson: Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Bloch:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties.

scholarly journals Novel Insights Into Systemic Iron Regulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-1-SCI-1
Author(s):  
Laura Silvestri ◽  
Alessia Pagani ◽  
Antonella Nai ◽  
Clara Camaschella
Keyword(s):  
Iron Deficiency ◽  
Iron Overload ◽  
Transferrin Receptor ◽  
Hereditary Hemochromatosis ◽  
Deficiency Anemia ◽  
Type I ◽  
Mrna Levels ◽  
Iron Availability ◽  
Smad Pathway ◽  
Hepcidin Expression

Abstract Iron, an essential element in mammals, is absorbed by duodenal enterocytes, enters the circulation through the iron exporter ferroportin, (FPN), circulates bound to transferrin and is uptaken through Transferrin Receptor 1. If in excess, iron is stored in macrophages and hepatocytes and released when needed. To maintain systemic iron homeostasis and to avoid the formation of "non transferrin bound iron" (NTBI), a highly reactive form which causes organ damage, the liver synthetizes hepcidin that, binding FPN, blocks iron export to the circulation. Hepcidin integrates signals from body iron, erythropoiesis and inflammatory cytokines. Defective hepcidin production causes iron overload and organ failure in Hereditary Hemochromatosis and Thalassemia; hepcidin excess leads to anemia in Iron Refractory iron Deficiency Anemia (IRIDA) and Anemia of Inflammation (AI). In hepatocytes hepcidin is under the control of the BMP-SMAD pathway, which is activated in a paracrine manner by BMP2 and BMP6 produced by liver sinusoidal endothelial cells. BMP2 maintains hepcidin basal levels, while BMP6 controls its expression in response to iron. The two ligands have different affinity for BMP type I receptors ALK2 and ALK3, suggesting two distinct branches of the hepcidin activation pathway. This possibility is consistent with the non-redundant function of BMP2 and BMP6, the different iron phenotype of hepatocyte-conditional ALK2 and ALK3 KO mice and the residual ability of BMP6 to activate hepcidin in hemochromatosis mice. Moreover ALK2, but not ALK3, is inhibited by the immunophilin FKBP12 in the absence of ligands. The BMP pathway activation depends upon the coreceptor hemojuvelin (HJV), the MHC class I protein HFE and the second transferrin receptor (TFR2). Mutations of all these proteins lead to decreased hepcidin expression in hemochromatosis. Hepcidin expression is inhibited in iron deficiency, hypoxia and when erythropoiesis is increased. Inhibitors are the liver transmembrane serine protease TMPRSS6, whose genetic inactivation causes IRIDA, and the erythroid hormone erythroferrone (ERFE), which is released by erythropoietin-stimulated erythroblasts. The mechanism of hepcidin inhibition by ERFE is unclear; still to allow ERFE function the BMP-SMAD pathway has not to be hyperactive. Intriguingly, both iron deficiency and erythropoiesis require epigenetic modifications at the hepcidin locus with HDAC3-dependent reversible loss of H3K9ac and H3K4me3. Hepcidin also acts as an antimicrobial peptide since its expression, increased by proinflammatory cytokines, such as IL6 through JAK2-STAT3 signaling, restricts iron availability for microbial growth. This first-line of defense against infections negatively influences erythropoiesis since chronic hepcidin activation causes AI. Despite persistent JAK2-STAT3 activation, inhibition of the BMP-SMAD pathway reduces hepcidin activation in AI experimental rodent models, suggesting that hepcidin activation in inflammation requires a functional BMP-SMAD pathway. Independently from hepcidin, inflammation also reduces FPN mRNA levels, favoring macrophage iron sequestration. The identification of hepcidin-ferroportin axis molecular players has translational implications. In primary and secondary iron overload hepcidin agonists (hepcidin peptides or mimics, agents that inhibit the hepcidin inhibitor TMPRSS6 and likely the ALK2-inhibitor FKBP12) and ferroportin inhibitors are potentially useful to prevent iron overload and/or to favor iron redistribution to macrophages. In case of AI, hepcidin antagonists (including anti-hepcidin, anti-HJV and anti-BMP6 monoclonal antibodies, L-enantiomeric oligonucleotides targeting hepcidin, siRNA against hepcidin, non-anticoagulant heparins, the ALK2 inhibitor momelotinib) might improve erythropoiesis increasing iron availability. The effect of some agents that have now entered the clinical phase will become apparent in the coming years. Disclosures Camaschella: vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals DISC-a, the First in a Novel Class of Potent and Selective Matriptase-2 Inhibitors for the Treatment of Hematologic Disorders Characterized By Low Hepcidin

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-42
Author(s):  
Vu Hong ◽  
Ravi Krishna Babu ◽  
Cecile Blaustein ◽  
Sophia Nguyen ◽  
Venkateshwar Rao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Iron Overload ◽  
Proteolytic Activity ◽  
Iron Homeostasis ◽  
Transferrin Saturation ◽  
Subcutaneous Administration ◽  
Hek293 Cells ◽  
Private Company ◽  
Hepcidin Expression ◽  
Hepcidin Gene

Hepcidin is known as the master regulator of systemic iron homeostasis with reduction in synthesis leading to the development of iron overload. Hepcidin gene expression is negatively modulated by matriptase-2 (MT-2), a liver-specific type II transmembrane serine protease. MT-2 cleaves hemojuvelin (HJV), leading to the extracellular release of soluble HJV fragments and suppression of hepcidin expression. Loss-of-function of MT-2 leads to increased hepcidin expression, as has been established by human genetics (Finberg et al., 2008) and genetic mouse models (Du et al., 2008). Therefore, inhibition of MT-2 represents a potential therapeutic strategy for diseases caused by inappropriately low hepcidin leading to iron overload or where therapeutic iron restriction may be used to control excessive erythrocytosis. Here we describe the characteristics of DISC-A, a potent (low nM Ki) small molecule MT-2 inhibitor for treatment of low hepcidin disorders, with a favorable pharmacokinetics profile in rats (Clp 6.4 ml/min/kg, and t ½ 4.6 hr) and monkeys (Clp 8.1 ml/min/kg, and t ½ 2.8 hr) and drug-like properties. DISC-A inhibits proteolytic activity of MT-2 expressed on the surface of transfected HEK293 cells and prevents shedding of MT-2 from the membrane (autocleavage). In addition, in MT-2 and HJV co-transfected HEK293A cells, DISC-A shows a dose dependent inhibition of HJV cleavage. The efficacy of DISC-A was evaluated in a rat model of low hepcidin. In this model, when Sprague-Dawley rats who are fed a standard iron diet (45 ppm) reach 8 - 9 weeks of age, they are administered erythropoietin (EPO) at 30 IU/animal/day for 4-consecutive days, before dosing with DISC-A. Under the conditions of the model, the increased erythropoiesis leads to increased iron utilization and consequently suppressed hepcidin levels. We determine hepcidin changes by measuring the changes in the expression of liver HAMP (the gene that encodes hepcidin) mRNA expression. Circulating soluble HJV is assayed as a direct measure of MT-2 activity. In this model, a single subcutaneous administration of DISC-A at 20 mg/kg resulted in a 50% reduction in soluble HJV, 14-fold increase in liver HAMP expression and &gt;50% reduction in serum iron and transferrin saturation (TSAT) at 2, 4, 6, and 8 hours. The pharmacokinetics/pharmacodynamics response was robust. In summary, we have identified DISC-A, a novel, potent inhibitor of MT-2. We have demonstrated that DISC-A inhibits MT-2 proteolytic activity, prevents cleavage of HJV, and modulates hepcidin gene expression and iron homeostasis in vitro and in vivo. The favorable pharmacokinetics suggest compounds from these chemical series have the potential for clinical therapeutic benefit. Disclosures Hong: Disc Medicine: Current Employment, Current equity holder in private company. Babu:Aurigene Discovery Technologies: Current Employment. Blaustein:Disc Medicine: Current Employment, Current equity holder in private company. Nguyen:Disc Medicine: Current Employment, Current equity holder in private company. Rao:Aurigene Discovery Technologies: Current Employment. Savage:Disc Medicine: Current Employment, Current equity holder in private company. MacDonald:Disc Medicine: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Beconi:Disc Medicine: Current Employment, Current equity holder in private company. Venkatraman:Disc Medicine: Current Employment, Current equity holder in private company.

scholarly journals Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5195-5204 ◽  
Author(s):  
Yin Xia ◽  
Jodie L. Babitt ◽  
Yisrael Sidis ◽  
Raymond T. Chung ◽  
Herbert Y. Lin
Keyword(s):  
Iron Metabolism ◽  
Human Liver ◽  
Bmp Signaling ◽  
Type I ◽  
Type Ii ◽  
Hepcidin Expression ◽  
Bmp Receptors ◽  
Distinct Subset ◽  
Morphogenetic Protein

Abstract Hemojuvelin (HJV) is a coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. However, the precise combinations of BMP ligands and receptors used by HJV remain unknown. HJV has also been demonstrated to bind to neogenin, but it is not known whether this interaction has a role in regulating hepcidin expression. In the present study, we show that BMP-2, BMP-4, and BMP-6 are endogenous ligands for HJV in hepatoma-derived cell lines, and that all 3 of these ligands are expressed in human liver. We demonstrate in vitro that HJV selectively uses the BMP type II receptors ActRIIA and BMPRII, but not ActRIIB, and HJV enhances utilization of ActRIIA by BMP-2 and BMP-4. Interestingly, ActRIIA is the predominant BMP type II receptor expressed in human liver. While HJV can use all 3 BMP type I receptors (ALK2, ALK3, and ALK6) in vitro, only ALK2 and ALK3 are detected in human liver. Finally, we show that HJV-induced BMP signaling and hepcidin expression are not altered by neogenin overexpression or by inhibition of endogenous neogenin expression. Thus, HJV-mediated BMP signaling and hepcidin regulation occur via a distinct subset of BMP ligands and BMP receptors, independently of neogenin.

Hepcidin Expression Is Regulated by a Complex of Hemochromatosis-Associated Proteins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 267-267 ◽  
Author(s):  
Paul J. Schmidt ◽  
Franklin W. Huang ◽  
Diedra M. Wrighting ◽  
Paul T. Toran ◽  
Nancy C. Andrews
Keyword(s):  
Transferrin Receptor ◽  
Iron Homeostasis ◽  
Hepatoma Cell ◽  
Bmp Signaling ◽  
Hepatoma Cell Line ◽  
Type I ◽  
Mutant Form ◽  
Iron Release ◽  
Hepcidin Expression ◽  
Bmp Receptors

Abstract Hemochromatosis is a common genetic disease resulting from increased dietary iron absorption and tissue iron deposition. Mutations in five unrelated genes are known to cause hemochromatosis in humans and mice. These encode the classic hemochromatosis protein (HFE), transferrin receptor 2 (TFR2), the iron exporter ferroportin (FPN), hemojuvelin (HJV), and the circulating anti-microbial peptide hepcidin (HAMP). Hepcidin binds to FPN, causing its internalization and degradation, thus decreasing cellular iron release. A basic understanding of the pathophysiology of FPN and hepcidin mutations has recently been elucidated; however, it was still unclear how mutations in HFE, TFR2, and HJV cause hemochromatosis. All are associated with decreased hepcidin production and inappropriately high levels of ferroportin activity. HFE, TFR2 and HJV are normally expressed in the hepatic cells that produce hepcidin. With collaborators, we showed that HJV acts as a bone morphogenetic protein (BMP) co-receptor. HJV binds to the BMP ligands and forms a complex with Type I BMP receptors, resulting in signaling through a SMAD pathway and induction of hepcidin expression. Disease causing mutations in HJV abrogate BMP co-receptor activity, and hepatocytes from Hjv−/ − mice have a blunted response to BMP2. HFE was known to form a complex with the classical transferrin receptor, TFR1. Several models have been proposed implicating this complex in the regulation of normal iron homeostasis, but they have not taken the role of hepcidin into account. To examine the HFE/TFR1 interaction in vivo, we developed mice expressing a mutant form of TFR1 that should constitutively interact with HFE. We found that these transgenic animals have a phenotype similar to Hfe−/ − mice, suggesting that TFR1 serves to sequester HFE to silence its activity. We next asked whether HFE might also participate in BMP signaling. We found that forced expression of HFE in a hepatoma cell line induces transcription of a reporter gene linked to the hepcidin promoter. It also induces transcription from a heterologous promoter containing BMP-responsive elements, suggesting that HFE works through the BMP pathway. In contrast, forced expression of TFR2 did not amplify expression of either reporter, but it prevented cellular release of a soluble cleavage product of HJV. Furthermore, we showed that both HFE and TFR2 are associated with HJV in a stable protein complex that can be isolated by co-immunoprecipitation or Ni-affinity chromatography. TFR2 appears to aid in the recruitment of HFE to this complex. We conclude that HFE and TFR2 thus serve to amplify BMP signaling through an HJV/BMP receptor pathway. Our findings provide a compelling explanation for the similar clinical hemochromatosis phenotypes resulting from mutations in these genes.

BMP/Smad signaling is not enhanced in Hfe-deficient mice despite increased Bmp6 expression

Blood ◽  
2009 ◽  
Vol 114 (12) ◽  
pp. 2515-2520 ◽  
Author(s):  
Léon Kautz ◽  
Delphine Meynard ◽  
Céline Besson-Fournier ◽  
Valérie Darnaud ◽  
Talal Al Saati ◽  
...  
Keyword(s):  
Iron Overload ◽  
Hereditary Hemochromatosis ◽  
Bmp Signaling ◽  
Signaling Cascade ◽  
Mrna Levels ◽  
Wild Type ◽  
Smad Signaling ◽  
Hepcidin Expression ◽  
Hepcidin Mrna ◽  
Deficient Mice

Abstract Impaired regulation of hepcidin expression in response to iron loading appears to be the pathogenic mechanism for hereditary hemochromatosis. Iron normally induces expression of the BMP6 ligand, which, in turn, activates the BMP/Smad signaling cascade directing hepcidin expression. The molecular function of the HFE protein, involved in the most common form of hereditary hemochromatosis, is still unknown. We have used Hfe-deficient mice of different genetic backgrounds to test whether HFE has a role in the signaling cascade induced by BMP6. At 7 weeks of age, these mice have accumulated iron in their liver and have increased Bmp6 mRNA and protein. However, in contrast to mice with secondary iron overload, levels of phosphorylated Smads 1/5/8 and of Id1 mRNA, both indicators of BMP signaling, are not significantly higher in the liver of these mice than in wild-type livers. As a consequence, hepcidin mRNA levels in Hfe-deficient mice are similar or marginally reduced, compared with 7-week-old wild-type mice. The inappropriately low levels of Id1 and hepcidin mRNA observed at weaning further suggest that Hfe deficiency triggers iron overload by impairing hepatic Bmp/Smad signaling. HFE therefore appears to facilitate signal transduction induced by the BMP6 ligand.

Aln-TMP: A Subcutaneously Administered RNAi Therapeutic Targeting Tmprss6 For The Treatment Of β-Thalassemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2260-2260 ◽  
Author(s):  
James Butler ◽  
Shannon Fishman ◽  
Tim Racie ◽  
Julia Hettinger ◽  
Brian R Bettencourt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Iron Overload ◽  
Serum Iron ◽  
Total Serum ◽  
Gene Suppression ◽  
Ineffective Erythropoiesis ◽  
Therapeutic Targeting ◽  
Hepcidin Expression ◽  
Secondary Iron Overload ◽  
Hepcidin Gene

Abstract The b-Thalassemias are a group of hereditary blood disorders resulting from insufficient beta globin production, ultimately giving rise to the signature clinical sequelae associated with β-Thalassemia which includes anemia, ineffective erythropoiesis, and secondary iron overload. Previously, we demonstrated that intravenous administration of an siRNA targeting hepatic Tmprss6 expression significantly ameliorated the disease phenotype in the Hbbth3/+ mouse model of Thalassemia Intermedia (Blood. 2013; 14;121(7):1200-8). The Tmprss6 gene encodes for the protein Matriptase-2 which negatively regulates Hepcidin gene expression by cleaving the Hepcidin regulatory protein Hemojuvelin; RNAi-mediated suppression of Tmprss6 removes this negative regulator, ultimately leading to an increase in Hepcidin expression. Increased Hepcidin expression leads to a significant decrease in serum iron concentration and Transferrin Saturation (TfSat), which in the β-Thalassemia disease setting, corrects ineffective erythropoiesis, ameliorates anemia, and mitigates secondary iron overload. The role of Tmprss6 in iron metabolism has been extensively characterized in animal and human studies and, together with the observation in the Hbbth3/+ mouse, represents an attractive therapeutic target for the treatment of β-Thalassemia. To this end, we developed ALN-TMP, a subcutaneous RNAi therapeutic targeting hepatic Tmprss6 for the treatment of β-Thalassemia. ALN-TMP employs the GalNAc conjugate siRNA delivery platform that safely and effectively delivers siRNA to the liver for hepatic gene silencing. Preclinical animal data demonstrate ALN-TMP exhibits robust and durable dose-dependent gene suppression as single dose administration of ALN-TMP leads to > 80% Tmprss6 gene suppression for up to 3 weeks post-dose. This leads to concomitant increases in Hepcidin gene expression (>2x baseline levels) and subsequent decreases in total serum iron and TfSat (>50% decrease from baseline). The degree to which ALN-TMP modulates Hepcidin and serum iron mobilization is nearly identical to that observed in the previous Hbbth3/+ mouse studies and suggests ALN-TMP is a potent RNAi therapeutic with the potential of producing disease modifying effects in β-Thalassemia. Disclosures: Butler: Alnylam: Employment. Fishman:Alnylam: Employment. Racie:Alnylam Pharmaceutical, Inc: Employment. Hettinger:Alnylam Pharmaceuticals: Employment. Bettencourt:Alnylam Pharmaceuticals: Employment. Charisse:Alnylam Pharmaceuticals: Employment. Fitzgerald:Alnylam: Employment.

Roles of BMP signaling and Nkx2.5 in patterning at the chick midgut-foregut boundary

Development ◽  
2000 ◽  
Vol 127 (17) ◽  
pp. 3671-3681 ◽  
Author(s):  
D.M. Smith ◽  
C. Nielsen ◽  
C.J. Tabin ◽  
D.J. Roberts
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Bmp Signaling ◽  
Pyloric Sphincter ◽  
Type I ◽  
Gut Development ◽  
Spatial And Temporal Patterns ◽  
Bmp Receptors ◽  
A Cell ◽  
Necessary And Sufficient

Patterning of the gut into morphologically distinct regions results from the appropriate factors being expressed in strict spatial and temporal patterns to assign cells their fates in development. Often, the boundaries of gene expression early in development correspond to delineations between different regions of the adult gut. For example, Bmp4 is expressed throughout the hindgut and midgut, but is not expressed in the early gizzard. Ectopic BMP4 in the gizzard caused a thinning of the muscularis. To understand this phenotype we examined the expression of the receptors transducing BMP signaling during gut development. We find that the BMP receptors are differentially expressed in distinct regions of the chicken embryonic gut. By using constitutively activated versions of the BMP type I receptors, we find that the BMP receptors act similarly to BMP4 in the gizzard when ectopically expressed. We show that the mesodermal thinning seen upon ectopic BMP signaling is due to an increase in apoptosis and a decrease in proliferation within the gizzard mesoderm. The mesodermal thinning is characterized by a disorganization and lack of differentiation of smooth muscle in the gizzard mesoderm. Further, ectopic BMP receptors cause an upregulation of Nkx2.5, the pyloric sphincter marker, similar to that seen with ectopic BMP4. This upregulation of Nkx2.5 is a cell-autonomous event within the mesoderm of the gizzard. We also find that Nkx2.5 is necessary and sufficient for establishing aspects of pyloric sphincter differentiation.

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