Twisted Gastrulation (TWSG1) Is Expressed at Elevated Levels in Thalassemia and Regulates Bone Morphogenic Protein Signaling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1785-1785
Author(s):  
Toshihiko Tanno ◽  
Prashanth Porayette ◽  
Ajoy Bhupatiraju ◽  
Pamela Staker ◽  
Y. Terry Lee ◽  
...  
Keyword(s):  
Mouse Model ◽  
Quantitative Pcr ◽  
Hepatoma Cell ◽  
Bone Morphogenic Protein ◽  
Bmp Signaling ◽  
Hepatoma Cell Line ◽  
Wild Type ◽  
Expression Levels ◽  
Hepcidin Expression ◽  
Twisted Gastrulation

Abstract Iron overload and bony abnormalities cause considerable morbidity among patients with thalassemia syndromes. One possible explanation for this phenomenon is that proteins normally secreted into the marrow microenvironment during erythropoiesis are over-expressed in thalassemia patients due to expanded and ineffective erythropoiesis. We previously discovered that GDF15 is produced at very high levels in thalassemia patients and inhibits hepcidin expression. Transcriptome screens of erythroblasts were utilized here to identify twisted gastrulation (TWSG1) as a second candidate protein for further study. Quantitative PCR using the β-thalassemia murine model (Hbbth3/+ β-thalassemia intermedia mouse model, n=13; Hbbth3/th3 β-thalassemia major mouse model, n=5) revealed that splenic expression levels of Tsg (murine TWSG1) were significantly higher in thalassemia mice (Hbbth3/+, 2.2E02 ± 2.7E01 copies/ng RNA, p<0.01; Hbbth3/th3, 5.3E02 ± 6.8E01 copies/ng RNA, p<0.01) than among wild type mice (4.7E01 ± 2.4E01 copies/ng RNA, n=7). Bone marrow expression of Tsg was elevated (Hbbth3/+, 1.1E02 ± 3.2E01 copies/ng RNA, p=0.17; Hbbth3/th3, 1.3E02 ± 2.2E01 copies/ng RNA, p<0.05) compared with wild type mice (5.3E01 ± 2.5E01 copies/ng RNA). Tsg expression levels in the murine liver were also significantly higher (Hbbth3/+, 2.8E02 ± 4.6E01 copies/ng RNA, p<0.05; Hbbth3/th3, 3.9E02 ± 4.9E01 copies/ng RNA, p<0.01) than in wild type mice (1.5E02 ± 4.0E01 copies/ng RNA). These results suggest that expression of Tsg is up-regulated in the murine β-thalassemia model. By comparison, murine Tsg expression was up-regulated to a greater extent than GDF15 in the thalassemia mice. In addition to murine studies, human studies of TWSG1 were performed. Quantitative PCR using cultured human CD34+ cells demonstrated the highest-level expression of TWSG1 at the early stages of erythroblast differentiation (9.3E02 ± 1.4E02 copies/ng RNA). Preliminary ELISA analyses demonstrated statistically significant elevations in TWSG1 levels in serum from thalassemia patients (n=18, 463 ± 41 ng/ml) when compared to serum from healthy volunteers (n=10, 310 ± 45 ng/ml, p<0.05), but the relative increase in TWSG1 in humans was far less than previously reported for GDF15. To determine whether TWSG1 regulates hepcidin expression, assays were performed using a human hepatoma cell line (HuH-7). Unlike GDF15, TWSG1 did not directly affect hepcidin expression as measured by quantitative PCR in dosed assays (1–1,000 ng/ml TWSG1). However, TWSG1 was found to suppress hepcidin through an indirect mechanism involving bone morphogenic protein (BMP). BMPs regulate several tissue-specific processes including bone remodeling and induction of hepcidin expression in liver cells. In dosed-titrations, ≥100 ng/ml of TWSG1 resulted in a 50% reduction (p<0.05) in the BMP2 augmentation of hepcidin expression. These novel data suggest that TWSG1 is expressed at elevated levels in thalassemia and has the potential to affect BMP signaling processes including the regulation of hepcidin.

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.

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.

scholarly journals The expression profile of miR-3099 during neural development of Ts1Cje mouse model of Down syndrome

2021 ◽  
Vol 4 (1) ◽  
pp. 7-15
Author(s):  
Shahidee Zainal Abidin ◽  
Han-Chung Lee ◽  
Syahril Abdullah ◽  
Norshariza Nordin ◽  
Pike-See Cheah ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Mouse Model ◽  
Neural Development ◽  
Progenitor Cell ◽  
Neuronal Cell ◽  
Expression Level ◽  
Wild Type ◽  
Nestin Expression ◽  
Cell Markers ◽  
Expression Levels

MicroRNA-3099 (miR-3099) plays a crucial role in regulating neuronal differentiation and development of the central nervous system (CNS). The miR-3099 is a pro-neuronal miRNA that promotes neural stem/progenitor cell (NSPC) differentiation into neuronal lineage by suppressing astrogliogenesis. Down syndrome (DS) brain exhibited increased astrogliogenesis and reduced neuronal cell density. The involvement of miR-3099 in the neurodevelopment of DS has not been investigated and potentially responsible for the neurogenic-to-gliogenic shift phenomenon observed in DS brain. To investigate the role of miR-3099 during DS brain development, neural/progenitor cell proliferation and differentiation, we profiled miR-3099 expression level in the Ts1Cje, a mouse model for DS. We analysed the Ts1Cje whole brain at embryonic day (E) 10.5, E14.5 and P1.5, proliferating neurospheres and differentiating neurospheres at 3, 9 and 15 days in vitro (DIV). Expression of miR-3099 in both the developing mouse brain and the differentiating neurosphere was not significantly different between Ts1Cje and wild type controls. In contrast, the expression level of miR-3099 was significantly higher (p<0.05) in proliferating NSPC derived from the Ts1Cje compared to wild-type. Further molecular profiling of NPSC and glial cell markers indicated that the expression of Sox2 (p<0.01) and Gfap (p<0.05) were significantly downregulated in Ts1Cje neurospheres as compared to that of wild type, respectively. While there were no significant differences in Tuj1 and Nestin expression levels between the Ts1Cje and wild type neurospheres, their expression levels were ~3-fold upregulated and ~2.6 downregulated Ts1Cje group, respectively. The findings suggest that dysregulation of miR-3099 affects NSPC lineage commitment as indicated by altered postmitotic neuronal cell markers. Further molecular characterisation and gene expression profiling of other neuronal and glial markers will help refine the analysis of gene-gene interactions underlying the neuropathologies of DS.

Erythroferrone Regulates Hepcidin Expression Independently of Matriptase 2

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3616-3616 ◽  
Author(s):  
Leon Kautz ◽  
Sharraya Aschemeyer ◽  
Victoria Gabayan ◽  
Tomas Ganz ◽  
Elizabeta Nemeth
Keyword(s):  
Limit Of Detection ◽  
Hematological Parameters ◽  
Isolated Hepatocytes ◽  
Bmp Signaling ◽  
The Body ◽  
Deficiency Anemia ◽  
Wild Type ◽  
Hepcidin Expression ◽  
Undetectable Plasma ◽  
Transmembrane Serine Protease

Abstract Introduction: The iron-regulatory hormone hepcidin regulates the body iron stores and its expression is repressed when erythropoietic activity intensifies to meet the iron requirements for erythropoiesis (e.g. during anemia). Under the influence of erythropoietin (EPO), the hormone erythroferrone (ERFE) is secreted by erythroid precursors in the bone marrow and the spleen, and suppresses hepcidin synthesis to facilitate the recovery from anemia. However, the mechanism by which ERFE suppresses hepcidin is unknown. In contrast with forms of anemia in which hepcidin is suppressed, patients with mutations in transmembrane serine protease 6 (TMPRSS6) have iron-refractory iron deficiency anemia (IRIDA) but increased hepcidin production despite a severe anemia and elevated EPO levels. Recently, it has been suggested that matriptase-2 activity facilitates ERFE-mediated suppression of hepcidin. We therefore investigated the potential crosstalk between ERFE and Matriptase 2. Methods: We first measured serum ERFE concentration in Tmprss6-/- mice. To assess the contribution of ERFE to the phenotype of Tmprss6-/-mice, we next generated Tmprss6-/-mice with disrupted Erfe (Erfe+/- Tmprss6-/-; Erfe-/- Tmprss6-/- and Erfe+/+ Tmprss6-/-). To determine whether ERFE requires TMPRSS6 to regulate hepcidin production, we treated freshly isolated hepatocytes from wild-type (WT) or Tmprss6-/- mice with conditioned medium from cells expressing recombinant ERFE or not. Results: While wild-type mice have undetectable plasma ERFE (below the 500 pg/ml limit of detection), plasma ERFE concentration was elevated in Tmprss6-/- to levels comparable to those of WT animals 24 hours after phlebotomy (~3 ng/ml) but was lower than ERFE levels in thalassemic mice (~10 ng/ml). Ablation of Erfe in Tmprss6-/- mice did not result in any change in hematological parameters, hepcidin expression and iron levels compared to Tmprss6-/- animals at 6 weeks of age. However, treatment of WT and Tmprss6-/-hepatocytes with ERFE resulted in a comparable suppression of hepcidin mRNA expression. Conclusion: Although matriptase-2 may dampen the BMP signaling under the influence of EPO, it is not part of the ERFE signaling pathway. Disclosures Ganz: Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor; Keryx Biopharmaceuticals: Consultancy. Nemeth:Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor.

scholarly journals Development of an interleukin (IL) 6 receptor antagonist that inhibits IL-6-dependent growth of human myeloma cells.

1994 ◽  
Vol 180 (6) ◽  
pp. 2395-2400 ◽  
Author(s):  
F D de Hon ◽  
M Ehlers ◽  
S Rose-John ◽  
S B Ebeling ◽  
H K Bos ◽  
...  
Keyword(s):  
Cell Line ◽  
Inflammatory Diseases ◽  
Hepatoma Cell ◽  
Myeloma Cell ◽  
Oncostatin M ◽  
Mutant Protein ◽  
Hepatoma Cell Line ◽  
Wild Type ◽  
Human Hepatoma Cell

The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)-transformed B cell line CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R) alpha complex with the signaltransducing "beta" chain, gp130, but leave the binding of IL-6 to IL-6R alpha intact. The resulting mutant protein (with substitutions of residues Gln160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL-6R alpha was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL-6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.

Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3

2007 ◽  
Vol 292 (1) ◽  
pp. G323-G328 ◽  
Author(s):  
S. F. Drake ◽  
E. H. Morgan ◽  
C. E. Herbison ◽  
R. Delima ◽  
R. M. Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Transferrin Receptor ◽  
Mutant Mouse ◽  
Hereditary Hemochromatosis ◽  
Mutant Mice ◽  
Wild Type ◽  
Hepcidin Expression ◽  
Fe Uptake ◽  
Type 3

Hereditary hemochromatosis type 3 is an iron (Fe)-overload disorder caused by mutations in transferrin receptor 2 (TfR2). TfR2 is expressed highly in the liver and regulates Fe metabolism. The aim of this study was to investigate duodenal Fe absorption and hepatic Fe uptake in a TfR2 (Y245X) mutant mouse model of hereditary hemochromatosis type 3. Duodenal Fe absorption and hepatic Fe uptake were measured in vivo by 59Fe-labeled ascorbate in TfR2 mutant mice, wild-type mice, and Fe-loaded wild-type mice (2% dietary carbonyl Fe). Gene expression was measured by real-time RT-PCR. Liver nonheme Fe concentration increased progressively with age in TfR2 mutant mice compared with wild-type mice. Fe absorption (both duodenal Fe uptake and transfer) was increased in TfR2 mutant mice compared with wild-type mice. Likewise, expression of genes participating in duodenal Fe uptake ( Dcytb, DMT1) and transfer (ferroportin) were increased in TfR2 mutant mice. Nearly all of the absorbed Fe was taken up rapidly by the liver. Despite hepatic Fe loading, hepcidin expression was decreased in TfR2 mutant mice compared with wild-type mice. Even when compared with Fe-loaded wild-type mice, TfR2 mutant mice had increased Fe absorption, increased duodenal Fe transport gene expression, increased liver Fe uptake, and decreased liver hepcidin expression. In conclusion, despite systemic Fe loading, Fe absorption and liver Fe uptake were increased in TfR2 mutant mice in association with decreased expression of hepcidin. These findings support a model in which TfR2 is a sensor of Fe status and regulates duodenal Fe absorption and liver Fe uptake.

scholarly journals Metabolic characteristics of a human hepatoma cell line stably transfected with hormone-sensitive lipase

1999 ◽  
Vol 341 (2) ◽  
pp. 453-460 ◽  
Author(s):  
Richard J. PEASE ◽  
David WIGGINS ◽  
E. David SAGGERSON ◽  
Jeni TREE ◽  
Geoffrey F. GIBBONS
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Hepatoma Cell ◽  
Hormone Sensitive Lipase ◽  
Acid Oxidation ◽  
Hepatoma Cell Line ◽  
Wild Type ◽  
Transfected Cells ◽  
Oxidative Pathway ◽  
Hormone Sensitive

Clones of HepG2 cells were selected that stably express the cDNA for hormone-sensitive lipase (HSL). When cells were cultured in the presence of labelled extracellular oleate, accumulation of labelled fatty acid as cellular triacylglycerol (TAG) was significantly lower in the transfectants compared with the wild-type cells. There was no change in the net rate of phospholipid (PL) synthesis. Culture of cells containing isotopically prelabelled TAG resulted in a greater net loss of TAG from the transfected cells than from the wild-type cells. The excess loss of labelled TAG was primarily due to an increased TAG fatty acid oxidation. Free fatty acid release into the medium was not increased in the transfectants, nor was the very low rate of lipoprotein lipid secretion. Also, there was no increased net trafficking of fatty acids from TAG into PLs. Changes in the 3H:14C ratio of TAG prelabelled with [3H]glycerol and [14C]oleate suggested that none of excess TAG fatty acid released in the transfected cells underwent intracellular re-esterification to TAG prior to oxidation. The results suggest that fatty acids mobilized by HSL are directed immediately into the oxidative pathway and are not available for biosynthetic processes. It appears likely, therefore, that intracellular TAG-derived fatty acids which enter the oxidative pathway exist in a different compartment from those that are directed towards synthesis.

scholarly journals Glucocorticoid-regulated glycoprotein maturation in wild-type and mutant rat cell lines.

1986 ◽  
Vol 103 (6) ◽  
pp. 2323-2331 ◽  
Author(s):  
G L Firestone ◽  
N J John ◽  
K R Yamamoto
Keyword(s):  
Cell Surface ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Wild Type ◽  
Glycoprotein Precursor ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Cell Surface Glycoproteins ◽  
Mutant Rat ◽  
Surface Glycoproteins

Glucocorticoid hormones can regulate the posttranslational maturation of mouse mammary tumor virus (MTV) precursor polyproteins in M1.54, a stably infected rat hepatoma cell line. We have used complement-mediated cytolysis to recover variants of M1.54 that fail to express MTV cell surface glycoproteins in a hormone-regulated manner (Firestone, G.L., and K.R. Yamamoto, 1983, Mol. Cell. Biol., 3:149-160). One such clonal isolate, CR4, is similar to wild-type with respect to synthesis of MTV mRNAs, production of the MTV glycoprotein precursor (gPr74env) and a glycosylated maturation product (gp51), and hormone-induced processing of two MTV phosphoproteins. In contrast, three viral cell surface glycoproteins (gp78, gp70, and gp32) and one extracellular species (gp70s), which derive from gPr74env in glucocorticoid-treated wild-type cells, fail to appear in CR4. CR4 showed no apparent alterations in proliferation rate, cell shape, or expression of total functional mRNA and bulk glycoproteins. We conclude that the genetic lesion in CR4 defines a highly selective hormone-regulated glycoprotein maturation pathway that alters the fate of a restricted subset of precursor species.

Discovery of Growth Differentiation Factor 15 as an Erythroblast-Secreted Regulator of Hepcidin with Very High Level Expression in Patients with Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 553-553 ◽  
Author(s):  
Toshihiko Tanno ◽  
Natarajan V. Bhanu ◽  
Patricia A. Oneal ◽  
Sung-Ho Goh ◽  
Pamela Staker ◽  
...  
Keyword(s):  
Iron Overload ◽  
Plasma Levels ◽  
Hepatoma Cell ◽  
Precursor Cell ◽  
Hepatoma Cell Line ◽  
High Level Expression ◽  
Growth Differentiation Factor 15 ◽  
Hepcidin Expression ◽  
Differentiation Factor ◽  
High Level

Abstract Iron overload causes considerable morbidity associated with thalassemia due to inappropriate suppression of the iron regulator, hepcidin. One possible explanation for this phenomenon is that protein(s) that are normally secreted into the marrow microenvironment by erythroblasts become endocrine signals in patients with thalassemia due to the myeloproliferative nature of the disease. To test this hypothesis, progenitor and precursor cell transcriptional profiles were generated with Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays using primary erythroblasts cultured from 15 healthy human donors. For this study, informatic analyses were focused upon 54 members of the TGF–B/BMP superfamily. Among the subset of genes identified by this screen, evidence for high-level expression of a gene named growth differentiation factor 15 (GDF15) was discovered in the precursor cell profiles. Quantitative PCR, Western, and ELISA analyses confirmed expression and secretion of GDF15 during erythroblast maturation. GDF15 is an apoptosis-associated protein expressed primarily by the placenta. To determine whether GDF15 serves as a regulator of hepcidin expression, hepcidin expression assays were performed using a human hepatoma cell line (HuH-7). BMP2 and BMP4 were studied for comparison. Addition of BMP2 and BMP4 (range 10,000–500,000 pg/ml) resulted in dosed increases in hepcidin mRNA (5–30 fold). At the concentrations of GDF15 normally found in human blood (500 pg/ml), a 2-fold increase in the expression of hepcidin was measured compared to matched cultures containing no supplemental GDF15. However, GDF15 dosed to levels above 5,000 pg/ml resulted in a significant reduction in hepcidin expression. Next, plasma levels of GDF15 were measured in the peripheral blood of 162 donors (donor groups included healthy controls, sickle-cell syndromes, thalassemia syndromes, and other causes of anemia) to determine whether plasma GDF15 levels are dysregulated in thalassemia. Plasma samples from 21 hereditary hemochromatosis donors provided evidence that significantly elevated GDF15 expression is not associated with iron overload in the absence of erythroid pathology. Compared with mean plasma GDF15 levels of 536±222 pg/ml among the control samples, only patients with thalassemia intermedia, thalassemia major, and HbE-beta thalassemia showed significantly elevated plasma levels of GDF15 (9 donors; mean GDF15 24,600 pg/ml; range 8,980–75,100 pg/ml; p<0.01). Elevated ferritin levels (>370 ug/L) were noted in each of those patients regardless of their transfusion or chelation history. These novel findings suggest that GDF15 is secreted from human erythroblasts, released into the circulation at extremely high levels in thalassemia patients, and contributes to iron overloading in those patients by suppressing hepcidin expression.

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