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 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.

Direct Upregulation of Iron Regulatory Hormone Hepcidin by Interferon Alpha

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2103-2103
Author(s):  
Kazuhiko Ichiki ◽  
Katsuya Ikuta ◽  
Hiroki Tanaka ◽  
Yusuke Sasaki ◽  
Yasushi Shimonaka ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Research Funding ◽  
Iron Absorption ◽  
Primary Hepatocytes ◽  
Hepcidin Expression ◽  
Ifn Alpha ◽  
Serum Hepcidin ◽  
Ifn Treatment ◽  
Hepcidin Gene ◽  
Hepcidin Mrna

Abstract Abstract 2103 Introduction: Interferon (IFN) activates various immune systems in vivo and is administered for the patients with various diseases such as viral hepatitis B,C and malignant tumors. During the treatment with IFN, iron metabolism has been reported to be important. For instances, iron overload has reported to contribute to the poor response rate of IFN treatment and poor prognosis in the patients with hepatitis C. However, it has been unclear whether IFN itself effects on the iron metabolism. Therefore, we aimed to determine the effect of IFN itself on iron metabolism. Materials and Methods: The mice were subcutaneously administered the mouse IFN alpha (104 and 105 IU/mouse/day) and sacrificed after 3, 5 and 7 days. Then the serum, the bone marrow, the liver, the spleen, and the duodenum tissues were collected. To analyze serum hepcidin levels, liquid chromatography-tandem mass spectrometry was performed. The changes of mRNA and protein expressions involved in iron metabolism such as transferrin receptor 1, hepcidin, and ferroportin, were also analyzed by quantitative real time RT-PCR and western blotting. Some parts of tissues were processed for the formalin-fixed paraffin embedding blocks, and Hematoxylin-Eosin and immunofluorescence staining for ferroportin. To evaluate the direct effects of IFN alpha on the expression of hepcidin by hepatocytes, primary cultured hepatocytes obtained from the mouse liver and human hepatoma-derived cultured cells were treated with human IFN alpha, and then RNA and the protein were extracted after 24 hours of treatment, followed by the analysis of hepcidin mRNA and transcription factors, such as STAT3. Results: Among the analyzed genes and proteins that are involved in iron metabolism, the hepcidin expression was highly modulated by IFN alpha. Both the serum hepcidin level and hepcidin mRNA expression in the liver were increased in IFN alpha-treated mice. And the expression of ferroportin, target molecule of hepcidin, was decreased in the duodenum of IFN alpha-treated mice. The analysis in vitro showed the upregulation of hepcidin mRNA by the IFNa treatment both in primary hepatocytes of the mouse liver and human hepatoma-derived cells. Interestingly, the upregulation of activated STAT3 was also observed in those cells, and that was cancelled by the addition of neutralizing antibody (IFNAR1) for IFN receptors. Discussion and Conclusion: Our results showed that IFN alpha directly upregulated the hepcidin expression by the liver. Concerning the mechanism of the upregulation of hepcidin, the upregulation of activated STAT3 by IFN should be involved. Commonly, IFN alpha accelerated the heterodimer formation of transcription of STAT1 and STAT2 via the activation of JAK by the binding of IFN alpha with IFN receptor on cell surface. The upstream lesion of the hepcidin gene does not have the binding site for STAT1/STAT2 heterodimer, but have the binding site for STAT3 homodimer. In addition, IFNa has been reported to activate STAT3 in human primary hepatocytes (Radaeva, S. et al. Gastroenterology 2002; 122:1020–34). Combined these, presumably IFN alpha directly effected on hepatocytes to enhance the transcription of hepcidin gene via activated STAT3 homodimer. In addition to the upregulation of hepcidin, our results showed the decreased duodenum expression of ferroportin in IFN alpha-treated mice. Ferroportin is expressed on the basolateral cell membrane of enterocytes and has function of iron transport from the enterocytes to blood. Ferroportin has also been reported to internalize and be degraded when hepcidin binds to ferroportin on the cell surface, resulting in the decrease of iron absorption from the gastrointestinal tract. Therefore, the upregulation of hepcidin by IFN alpha treatment observed in the present study should be physiologically functional, and this indicated that iron absorption should be decreased during IFN treatment. This effect is considered to be favorable because this would inhibit iron overload during IFN treatment and may increase the effect of IFN. Disclosures: Sasaki: Novartis Pharma K.K.: Research Funding. Kohgo:Novartis: Research Funding, Speakers Bureau; Chugai Roche: Research Funding; Asahikasei Kurare Medical: Research Funding; Sapporo Brewery: Research Funding; Kyorin Pharma: Research Funding.

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.

Autocrine formation of hepcidin induces iron retention in human monocytes

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2392-2399 ◽  
Author(s):  
Igor Theurl ◽  
Milan Theurl ◽  
Markus Seifert ◽  
Sabine Mair ◽  
Manfred Nairz ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Iron Homeostasis ◽  
Mrna Levels ◽  
Anemia Of Chronic Disease ◽  
Hepcidin Expression ◽  
Inflammatory Monocytes ◽  
Cellular Iron ◽  
Iron Sequestration ◽  
Iron Retention ◽  
Hepcidin Mrna

Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte-derived hepcidin exerts autocrine regulation toward cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within 3 hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients, monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments using a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalization and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this lipopolysaccharide-mediated effect. Using ferroportin mutation constructs, we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface. Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

Chronic intrauterine pulmonary hypertension decreases calcium-sensitive potassium channel mRNA expression

2000 ◽  
Vol 279 (5) ◽  
pp. L857-L862 ◽  
Author(s):  
David N. Cornfield ◽  
Ernesto R. Resnik ◽  
Jean M. Herron ◽  
Steven H. Abman
Keyword(s):  
Gene Expression ◽  
Pulmonary Hypertension ◽  
Mrna Expression ◽  
Internal Control ◽  
Vascular Reactivity ◽  
Ductus Arteriosus ◽  
Critical Role ◽  
Mrna Levels ◽  
Channel Gene ◽  
Mrna Content

Calcium-sensitive potassium (KCa) channels play a critical role in mediating perinatal pulmonary vasodilation. Because infants with persistent pulmonary hypertension of the newborn (PPHN) have blunted vasodilator responses to birth-related stimuli, we hypothesized that lung KCachannel gene expression is decreased in PPHN. To test this hypothesis, we measured KCa channel gene expression in distal lung homogenates from both fetal lambs with severe pulmonary hypertension caused by prolonged compression of the ductus arteriosus and age-matched, sham-operated animals (controls). After at least 9 days of compression of the ductus arteriosus, fetal lambs were killed. To determine lung KCa channel mRNA levels, primers were designed against the known sequence of the KCa channel and used in semiquantitative RT-PCR, with lung 18S rRNA content as an internal control. Compared to that in control lambs, lung KCa channel mRNA content in the PPHN group was reduced by 26 ± 6% ( P < 0.02), whereas lung voltage-gated K+ 2.1 mRNA content was unchanged. We conclude that lung KCa channel mRNA expression is decreased in an ovine model of PPHN. Decreased KCa channel gene expression may contribute to the abnormal pulmonary vascular reactivity associated with PPHN.

scholarly journals The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Alexandria A. Reinhart ◽  
Angela T. Nguyen ◽  
Luke K. Brewer ◽  
Justin Bevere ◽  
Jace W. Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Small Rnas ◽  
Iron Homeostasis ◽  
Critical Role ◽  
Opportunistic Pathogen ◽  
Lung Infection ◽  
Content Type ◽  
The Individual

ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrHIG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrHIG sequence [prrF(ΔHIG)]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.

Sensing Iron: Reciprocal Regulation of Hepcidin mRNA by Soluble and Cell-Associated Hemojuvelin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 512-512
Author(s):  
Lan Lin ◽  
Y. Paul Goldberg ◽  
Tomas Ganz
Keyword(s):  
Iron Homeostasis ◽  
Genomic Sequence ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Hepatoma Cell ◽  
Hepcidin Expression ◽  
Juvenile Hemochromatosis ◽  
Hepcidin Mrna

Abstract Human genetic studies identified HJV (also called HFE2) as the major cause for juvenile hemochromatosis (JH). Patients with HJV hemochromatosis have low urinary levels of hepcidin, the principal iron-regulatory hormone secreted by the liver. We attempted to establish the specific roles of HJV in iron metabolism, especially its relationship with hepcidin. Translation of the genomic sequence indicated a C-terminal GPI anchor for the protein product of HJV, hemojuvelin. This suggested that hemojuvelin may have either a soluble or a cell-associated form. In human hepatoma cell line Hep3B, knockdown of cellular HJV by siRNA decreased hepcidin expression, independently of the IL-6 pathway. Intriguingly, the addition of recombinant soluble hemojuvelin (rs-hemojuvelin) also suppressed hepcidin expression in primary human hepatocytes, in a log-linear dose-dependent manner, suggesting competition between soluble and cell-associated forms of hemojuvelin. Soluble hemojuvelin was found in human sera at concentrations similar to those required to suppress hepcidin mRNA in vitro. In cells engineered to express hemojuvelin, soluble hemojuvelin release was progressively inhibited by increasing iron or holotransferrin concentrations. Our study suggests that soluble and cell-associated hemojuvelin reciprocally regulate hepcidin mRNA levels, and that hemojuvelin may serve as a molecular messenger for iron homeostasis. Even in hepatocytes stimulated with IL-6, we observed strong suppression of hepcidin mRNA by rs-hemojuvelin. If rs-hemojuvelin or its active fragments also suppress hepcidin production in vivo, they could be used to alleviate anemia of inflammation.

The Mask Mutation Identifies TMPRSS6 as an Essential Suppressor of Hepcidin Gene Expression, Required for Normal Uptake of Dietary Iron.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3-3 ◽  
Author(s):  
Ernest Beutler ◽  
Pauline Lee ◽  
Terri Gelbart ◽  
Xin Du ◽  
Bruce Beutler
Keyword(s):  
Iron Deficiency ◽  
Serine Protease ◽  
Positional Cloning ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Dietary Iron ◽  
Iron Deficient ◽  
Total Body ◽  
Hepcidin Gene ◽  
Hepcidin Mrna

Abstract Hepcidin, the central negative regulator of iron absorption and iron release from macrophages, is upregulated by iron. Mutations in hemojuvelin, Hfe, transferrin receptor 2, and SMAD4 are known to prevent upregulation. Additionally, the bone morphogenetic proteins (BMPs), and the inflammatory cytokines IL-1 and IL-6 stimulate hepcidin gene activation. Downregulation of hepcidin is effected by anemia and hypoxia, but nothing is known of the mechanism through which this occurs. Here we describe the recessive ENU-induced phenotype Mask, so called because affected homozygotes developed regional alopecia in which truncal hair was shed while facial hair was retained. The Mask phenotype was found to be a manifestation of iron deficiency, and was eliminated by correcting the iron deficiency. When fed an iron deficient diet, mutant mice absorbed less iron than controls, as measured by total body 59Fe counting. After reaching a plateau total body counts stabilized, indicating that blood loss did not play a role in the iron deficiency. The level of liver hepcidin mRNA of iron deficient mice is normally greatly decreased; in contrast, the Mask mouse had high liver hepcidin mRNA levels. By positional cloning, we were able to ascribe the Mask phenotype to a splicing error in the Tmprss6 gene, which encodes a membrane-bound serine protease of previously unknown function. The mutation truncates the protein, eliminating the serine protease domain. Transfecting HepG2 cells to express the wildtype TMPRSS6 protein decreased baseline hepcidin reporter activity and almost entirely blunted the hepcidin inducing effect of IL-6, IL-1, hemojuvelin, and the BMPs. A construct encoding the Mask truncation mutant had diminished activity. Thus, TMPRSS6 powerfully down-regulates hepcidin gene transcription in the baseline state and prevents its upregulation by all known stimulators. TMPRSS6 is a non-redundant component of a hepcidin suppression pathway that exerts dominant effect over all known hepcidin inducing pathways, and is required for normal absorption of dietary iron.

Effect of High Levels of Growth Differentiation Factor 15 (GDF15) On Hepcidin Expression in Monocytes of β-Thalassemia Intermedia Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4061-4061
Author(s):  
Kleber Yotsumoto Fertrin ◽  
Carolina Lanaro ◽  
Carla Fernanda Franco-Penteado ◽  
Dulcinéia M Albuquerque ◽  
Mariana R. B. Mello ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mononuclear Cells ◽  
Positive Control ◽  
Beta Thalassemia ◽  
Iron Loading ◽  
Thalassemia Intermedia ◽  
Control Groups ◽  
Hepcidin Expression ◽  
Healthy Control ◽  
Hepcidin Gene

Abstract Abstract 4061 Poster Board III-996 Ineffective erythropoiesis in thalassemia has been associated with inappropriate suppression of hepatic synthesis of the key iron regulatory peptide hepcidin, leading to spontaneous iron overload. Hepcidin mRNA was found to be suppressed in hepatocyte cultures by high levels of growth differentiation factor 15 (GDF15) detected in sera from patients with thalassemic syndromes. GDF15 may inhibit hepcidin production by antagonizing positive regulatory cytokines such as bone morphogenic protein 6 (BMP6), shown to stimulate hepatic hepcidin expression in mouse models. Although mainly produced in the liver, human hepcidin production occurs to a lesser extent in circulating monocytes. Studies with monocytes in patients with anemia of chronic disease showed increased hepcidin expression and iron retention, but to the best of our knowledge, monocyte-derived hepcidin has not yet been characterized in iron-loading anemias such as beta-thalassemia intermedia (TI). We evaluated GDF15 plasmatic levels and correlated these to hemoglobin (Hb) levels, reticulocyte counts and gene expressions in monocytes from transfusion-independent, non-chelated TI patients, homozygous for the IVS-I-6 T→C mutation (n=18), healthy, age-matched controls with no iron deficiency or overload (n=10) and transfusion-independent sickle cell anemia (SCA) patients in steady state (n=5), as a positive control group in which hyperexpression of hepcidin in mononuclear cells has been previously demonstrated. Total RNA was extracted from monocytes isolated from peripheral blood mononuclear cells and determination of BMP6 and hepcidin gene expression was performed by Real Time Polymerase Chain Reaction. Plasma GDF15 levels were determined by ELISA. Mean TI patient Hb and serum ferritin levels were 7.05±0.21g/dL and 1846±346.4μg/L, respectively. Mean absolute reticulocyte count in TI was 163.9±21.5×103/mm3. Mean GDF15 plasma levels differences were statistically significant among TI, SCA and healthy control groups (8390±827, 1780±460 and 196±21pg/mL, p<0.0001, respectively). Hepcidin gene expression did not differ significantly between TI and healthy control groups (0.007±0.006 vs. 0.05±0.03, p>0.05, respectively) but was elevated in our positive control SCA patient group (0.56±0.20; p=0.04). BMP6 gene expression was significantly decreased in TI patients compared to healthy controls (1.17±0.15 vs. 0.51±0.11, p=0.01, respectively). GDF15 concentrations correlated positively with reticulocyte counts (r=0.47; p=0.007) and negatively with hemoglobin levels (r=-0.74; p<0.0001) and BMP6 gene expression (r=-0.62; p=0.006). Our data show very high GDF15 plasma levels in a relatively homogenous population of patients with iron overload secondary to beta-thalassemia intermedia. Correlation of GDF15 with hematimetric parameters reinforces its relation to the degree of erythropoietic activity in beta thalassemia due to ineffective erythropoiesis. In addition, our study demonstrates that monocyte-derived hepcidin, like its hepatic counterpart, is inappropriately suppressed in iron-overloaded beta thalassemia intermedia patients in the presence of increased GDF15 production, correlated to decreased levels of BMP6 expression. This supports the possibility that GDF15/BMP interaction regulates hepcidin production in monocytes and hepatocytes in a similar manner, and further studies of monocyte-derived hepcidin regulation may prove to be a suitable, non-invasive tool for the investigation of human liver-derived hepcidin pathways in thalassemia and other iron-loading anemias. Disclosures: No relevant conflicts of interest to declare.

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