Obligate N-Terminal but Not C-Terminal Monoferric Transferrin Ameliorates Anemia in β-Thalassemic Mice

Abstract Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe (monoferric N-TF), the C-lobe (monoferric-C), or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric. The iron-bound TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. Iron-bound TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and bone cells. Whereas TFR1 serves primarily as a cargo receptor, TFR2 serves primarily to influence cellular signaling events regulating hepcidin expression, erythropoiesis, and bone formation. We proposed that different transferrin forms provide differential signaling properties in this regulation. We thus generated TF mutant mice in which all iron-containing TF was either monoferric N (Tf monoN) or monoferric C (Tf monoC). Compared with Tf monoC mice, the Tf monoN mice demonstrated increased RBC production and increased hepcidin expression relative to iron status (Parrow et al. Blood). Based on observations in β-thalassemic mice treated with exogenous TF (Li et al. Nat Med), we hypothesized that β-thalassemic mice obligate for monoN TF would demonstrate improved erythropoietic and iron parameters compared with β-thalassemic mice obligate for monoC TF. To address this hypothesis, we crossed Hbb th3/+ mice (a mouse model of β-thalassemia intermedia) with Tf monoN and Tf monoC mice. Compared with Hbb th3Tf +/+mice, in Hbb th3/+Tf monoN mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology (Figure 1A-B), decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of ineffective erythropoiesis (as measured by the ratio of progenitors to RBC in the bone marrow). Additionally, serum ERFE was significantly reduced and hepcidin levels were increased in Hbb th3/+Tf monoN relative to Hbb th3/+Tf +/+controls. Conversely, hematological parameters from Hbb th3/+Tf monoC mice were comparable to Hbb th3/+Tf +/+ mice. Similarly, Hbb th3/+Tf monoCmice had no improvements in markers of ineffective erythropoiesis in the bone marrow compared with Hbb th3/+Tf +/+ mice. In summary, we demonstrate that the differential regulatory effects of monoN and monoC TF on erythropoiesis are relevant not only in steady-state, but also in the ineffective erythropoiesis that is characteristic of β-thalassemia. Because both monoN and monoC TF forms can deliver only one iron atom per TF-TFR1 binding event, our findings suggest that the improvements observed only in the Hbb th3/+Tf monoN mice were not due to iron restriction alone. We are now elucidating the mechanisms by which the two TF lobes exert their differential effects on ineffective erythropoiesis and exploring the translational potential of obligate monoN TF in the treatment of β-thalassemia. Figure 1 Figure 1. Disclosures Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

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

Blood ◽

10.1182/blood.v122.21.177.177 ◽

2013 ◽

Vol 122 (21) ◽

pp. 177-177

Author(s):

Laura Silvestri ◽

Marco Rausa ◽

Antonella Nai ◽

Alessia Pagani ◽

Clara Camaschella

Keyword(s):

Gene Expression ◽

Iron Status ◽

Cell Types ◽

Hepatic Iron ◽

Iron Dextran ◽

Wild Type ◽

Hepcidin Expression ◽

Cellular Iron ◽

Iron Deficient ◽

Parenchymal Cells

Abstract Introduction Iron-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. Methods Mice 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). Results In 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. Conclusions In 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.

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The Red Cell: How Erythropoiesis Modulates Hepcidin Expression

Blood ◽

10.1182/blood.v120.21.sci-24.sci-24 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-24-SCI-24

Author(s):

Yelena Ginzburg

Keyword(s):

Bone Marrow ◽

Iron Overload ◽

Iron Metabolism ◽

Conflicts Of Interest ◽

Peptide Hormone ◽

Well Being ◽

Iron Loading ◽

Ineffective Erythropoiesis ◽

Hepcidin Expression ◽

Erythroid Precursors

Abstract Abstract SCI-24 Erythroid precursors in the bone marrow require transferrin-bound iron for hemoglobin synthesis. Therefore, it is not surprising that the regulation of erythropoiesis and iron metabolism is interlinked. Iron demand for erythropoiesis is communicated to the iron-regulatory machinery through incompletely understood mechanisms. At the core of systemic iron homeostasis is the peptide hormone hepcidin, restricting cellular iron export to plasma by inducing the endocytosis and proteolysis of ferroportin. Hepcidin, a liver-synthesized peptide hormone, is released in response to increased iron load, and there is early evidence that circulating hepcidin concentrations affect the distribution of iron between the macrophage storage compartment (favored by higher hepcidin concentrations) and parenchymal cells, including cardiac myocytes and hepatocytes (favored by low hepcidin). Furthermore, ferroportin has recently been identified on erythroid precursors. Its purpose in this cell type and its function in the interface between erythropoiesis and iron metabolism are unclear. Additionally, in response to bleeding or the administration of erythropoietin, expansion of erythroid precursors suppresses hepcidin, most likely through one or more mediators released by the bone marrow and acting on hepatocytes. Iron-loading anemias with ineffective erythropoiesis, in particular β-thalassemia, demonstrate the effects of pathological “erythroid regulators” of hepcidin. Although erythrocyte transfusions are the main cause of iron loading in patients who receive them (β-thalassemia major), lethal iron overload is seen also in patients who are rarely or never transfused (β-thalassemia intermedia). Here, iron hyperabsorption is the cause of iron overload and, as in hereditary hemochromatosis, is caused by low hepcidin. Decreased hepcidin expression in β-thalassemia, with concurrent ineffective erythropoiesis and iron overload, indicates that the “erythroid regulator” may play an even more substantial role in iron metabolism than the “stores regulator.” Two members of the bone morphogenetic protein (BMP) family, growth differentiation factor (GDF) 15 and Twisted Gastrulation (TWSG1), have been implicated as candidate bone marrow-derived hepcidin suppressors in β-thalassemia. Neither factor is responsible for the physiologic hepcidin suppression in response to hemorrhage-induced stress erythropoiesis, and the physiologic suppressor is not known. We focus here on the current state of knowledge regarding the regulation of iron metabolism and attempt to elucidate the interface between iron regulation and erythropoiesis using evidence in part derived from animal models of β-thalassemia. A more complete understanding of the coregulation of erythropoiesis and iron metabolism may lay the foundation for improving diagnosis, increasing treatment options, and ultimately impacting the well-being of patients afflicted with different anemias and/or iron overload related-disorders. Disclosures: No relevant conflicts of interest to declare.

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Additive Effects of Decreased TfR1 and Ablated Erfe Improve Both Ineffective Erythropoiesis and Iron Overload in β-Thalassemic Mice

Blood ◽

10.1182/blood-2018-99-119426 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 847-847

Author(s):

Marc Ruiz Martinez ◽

Wenbin An ◽

Maria Feola ◽

Tomas Ganz ◽

Yelena Ginzburg

Keyword(s):

Bone Marrow ◽

Iron Overload ◽

Research Funding ◽

Iron Absorption ◽

Additive Effects ◽

Ineffective Erythropoiesis ◽

Triple Mutant ◽

Hepcidin Expression ◽

Equity Ownership ◽

La Jolla

Abstract Patients with β-thalassemias manifest anemia, ineffective erythropoiesis, extramedullary hematopoiesis, splenomegaly, and systemic iron overload. Even in non-transfusion dependent patients, iron overload in β-thalassemia develops because of increased intestinal iron absorption, leading to multiple organ dysfunction if untreated and accounts for most of the deaths in this disease. The main regulator of body iron content and distribution is hepcidin, inhibiting iron absorption in duodenal enterocytes and release of stored iron from macrophages and hepatocytes. Despite iron overload in patients and mice with β-thalassemia, hepcidin levels are insufficiently increased, as ineffective erythropoiesis dominates hepcidin regulation. Relatively low hepcidin causes iron overload in β-thalassemia. Recent evidence demonstrates that erythroferrone (ERFE), an erythroid regulator of hepcidin, is increased in bone marrow and serum from β-thalassemic patients and th3/+ mice [Kautz Nat Gen 2014] and its loss results in increased hepcidin, partially reversing iron overload in th3/+ mice [Kautz Blood 2015]. In addition, bone marrow ERFE expression normalizes in TfR1 haploinsufficient th3/+ mice [Li Blood 2017]. We hypothesize that the loss of ERFE and TfR1 influences erythropoiesis and iron metabolism in complementary ways in th3/+ mice, and therefore aim to explore iron- and erythropoiesis-related parameters in th3/+ TfR1+/- ERFE-/- (triple mutant (TM)) mice. All models are on a C57BL6 background and have been crossed to generate 4-6 mice for analysis at 6 weeks of age. We confirm our previous reports [Li Blood 2017] that th3/+TfR1+/- mice have increased RBC count and hemoglobin, decreased MCV and reticulocyte count (Table I), and reduce splenomegaly (Fig 1a and 1b) relative to th3/+ mice. We also confirm that th3/+ ERFE-/- mice do not reverse splenomegaly or improve peripheral blood circulating erythroid parameters compared to th3/+ mice [Kautz Blood 2015] (Table I) but exhibit further increase in TfR1 in late stage erythroid precursors (Fig 1c). Analysis of the bone marrow reveals that total erythroid mass is unaltered in triple mutants relative to th3/+, th3/+ ERFE-/-, and th3/+ TfR1+/- mice, but the number of late erythroblasts (poly-E and ortho-E stages) is normalized to WT levels (Fig 1d), strongly suggesting that, unlike in th3/+ erythropoiesis, where the block in differentiation occurs at the poly-E stage, th3/+ TfR1+/- and especially triple mutant mice restore differentiation at this stage to generate a higher hemoglobin. No differences in erythroblast apoptosis or ROS concentration are evident in triple mutant relative to th3/+ ERFE-/- or th3/+ TfR1+/- mice. We also analyzed markers of Epo responsiveness and demonstrate that serum Epo and EpoR expression are increased in th3/+ relative to WT mice (Fig 1e and 1f), but while serum Epo is decreased, EpoR is further increased (Fig 1f). These findings suggest that Epo responsiveness is more optimized in triple mutant erythroblasts, enabling a smaller proportion of late stage erythroblasts to produce circulating RBCs with relatively less serum Epo. Remarkably, while neither th3/+ ERFE-/- and th3/+ TfR1+/- mice reverse iron overload or impact hepcidin expression at 6 weeks of age, triple mutant mice demonstrate fully normalized ratio of hepcidin expression relative to liver iron concentration (LIC) (Fig 1g). Taken together, these experiments provide evidence of the differential and additive effects of TfR1 and ERFE loss in th3/+ mice, with a predominantly erythropoietic benefit of TfR1 loss, a predominantly iron-homeostatic benefit of ERFE loss, and synergy of both in optimizing Epo responsiveness. Disclosures Ganz: Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Silarus Pharma: Consultancy, Equity Ownership; Keryx Pharma: Consultancy, Research Funding; Gilead: Consultancy; Ablynx: Consultancy; Vifor: Consultancy; Akebia: Consultancy, Research Funding; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA.

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Increased Hepcidin Expression in β-Thalassemic Mice Treated with Apo-Transferrin Is Associated with Increased Smad1/5/8 and Decreased Erk1/2 Pathway Activation

Blood ◽

10.1182/blood.v124.21.747.747 ◽

2014 ◽

Vol 124 (21) ◽

pp. 747-747

Author(s):

Huiyong Chen ◽

Tenzin Choesang ◽

Petra Pham ◽

Weili Bao ◽

Maria Feola ◽

...

Keyword(s):

Bone Marrow ◽

Iron Overload ◽

Mrna Expression ◽

Fold Increase ◽

Isolated Hepatocytes ◽

Primary Hepatocytes ◽

Ineffective Erythropoiesis ◽

Hepcidin Expression ◽

Pathway Activation

Abstract Iron overload causes morbidity and mortality in patients with β-thalassemia. Transfusion independent patients develop iron overload from increased dietary iron absorption, implicating inappropriately low hepcidin and supporting the therapeutic potential of approaches to increase hepcidin. Relatively low liver hepcidin mRNA expression is also characteristic of mouse models of β-thalassemia, and a recently identified erythroid factor, erythroferrone, has been implicated in hepcidin suppression in thalassemia. We have previously shown that exogenous apo-transferrin injections result in relatively iron restricted erythropoiesis, ameliorate ineffective erythropoiesis, and increase hepcidin expression in Hbbth1/th1 β-thalassemia intermedia (thalassemic) mice. We now explore the effect of exogenous apo-transferrin on signaling pathways (i.e. Smad and Erk) and circulating parameters thought to participate in hepcidin regulation in vivo and in vitro in wild type (WT) and thalassemic mice. Our results demonstrate that apo-transferrin injection increase both serum hepcidin concentration (603 vs. 306 ng/ml, n=13-24 per group, P<0.0001) and liver mRNA expression (2.3-fold, n=8-12 per group, P=0.003) despite decreased circulating serum iron (96 vs. 180 μg/ml, n=6-8 per group, P=0.001) and parenchymal liver iron (1.0 vs. 1.3 µg/mg, n=10-12 per group, P=0.04) concentrations. Increased hepcidin in apo-transferrin treated thalassemic mice is unrelated to changes in liver Bmp6 mRNA expression (1.1-fold increase, n=8 per group, P=0.49) but correlates well with serum BMP2 concentration (1.3-fold increase, n=6-9 per group, P=0.03). Freshly isolated hepatocytes from thalassemic mice exhibit more pErk1/2 and a higher ratio of pErk1/2:Erk1/2 relative to WT mice, and apo-transferrin treated thalassemic mice exhibit a significant suppression of the Erk1/2 pathway (Figure 1) and increased Smad activation (1.4-fold increase, n=4-6 per group, P=0.02). These findings strongly suggest an inhibitory effect of the Erk pathway on hepcidin expression. We thus evaluate the effect of Erk inhibitor U0126 on freshly isolated hepatocytes from WT mice and demonstrate that erk inhibition in vitro also results in a dose-dependent increase in hepcidin expression (3.7-fold increase at 50µM U0126, P=0.0003) with no change in Smad1/5/8 phosphorylation. To further evaluate the role of circulating factors on the regulation of hepcidin expression with apo-transferrin treatment, we analyzed the effect of serum from PBS- and apo-transferrin treated WT and thalassemic mice on cultured primary hepatocytes from WT mice. A significant increase in hepcidin expression is observed in cells exposed to serum relative to untreated cells (Figure 2), while hepatocytes treated with serum from thalassemic mice demonstrate suppressed hepcidin expression and pSmad1/5/8 relative to WT serum (Figure 2 and 3). Furthermore, primary hepatocytes concurrently treated with serum and neutralizing anti-BMP2/4 antibodies have relatively suppressed hepcidin expression in each condition relative to cells treated with serum alone (Figure 2), suggesting again the importance of BMP2 in hepcidin expression. The treatment with serum and neutralizing anti-BMP2/4 antibodies also suppressed Smad1/5/8 and induces Erk1/2 pathway activation (Figure 3). Lastly, erythroferrone expression is increased in sorted orthochromatophilic bone marrow erythroblasts in thalassemic relative to WT mice and normalized by apo-transferrin injection in thalassemic mice (2.4-fold increase thalassemic vs. WT mice (P=0.008); 2-fold decrease apoTf-treated vs. PBS-treated thalassemic mice (P=0.03)). No differences are observed either in GDF15 or TWSG1 in sorted bone marrow orthochromatophilic erythroblasts. These findings support the importance of erythroferrone as an erythroid regulator in thalassemic mice, suggest that the effect in Hbbth1/th1 mice and Hbbth3/+ mice are comparable, and provides further evidence that treatment with apo-transferrin reverses ineffective erythropoiesis in thalassemic mice. In total, our findings support a model in which treatment of Hbbth1/th1 mice with apo-transferrin decreases bone marrow erythroferrone expression, decreases hepatocellular Erk activation, and increases Smad activation to increase liver hepcidin expression. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Westerman: Intrinsic Lifesciences LTD: Employment, Equity Ownership.

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Dual Deficiency of mDia1 and Mir-146a in an Age-Related Inflammatory Bone Marrow Microenvironment Induces Ineffective Erythropoiesis in Del(5q) MDS

Blood ◽

10.1182/blood.v128.22.3146.3146 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3146-3146

Author(s):

Yang Mei ◽

Ashley Basiorka ◽

Baobing Zhao ◽

Jing Yang ◽

Alan F List ◽

...

Keyword(s):

Bone Marrow ◽

Knockout Mice ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Ineffective Erythropoiesis ◽

Double Knockout ◽

Hepcidin Expression ◽

Molecular Abnormalities ◽

Molecular Pattern ◽

Age Related

Abstract Myelodysplastic syndromes (MDS) are a group of age-related clonal hematologic diseases characterized by anemia, neutropenia, and thromobocytopenia. The mechanisms of anemia in MDS are unclear, which is partially due to the heterogeneity of MDS involving many cytogenetic and molecular abnormalities. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, two genes located at chromosome 5q that is commonly deleted in MDS, causes an aged-related anemia and ineffective erythropoiesis that closely mimics human MDS. Bone marrow erythropoiesis at various stages was dramatically affected in mDia1/miR-146a double knockout mice, which induced a massive splenomegaly with potent extramedullary erythropoiesis. Old (> 1 year of age), but not young (2-4 months of age), wild type recipient mice that were transplanted with bone marrow cells from mDia1/miR-146a double knockout mice, exhibited severe anemia and rapid lethality, which indicates that the aged microenvironment is important for the development of ineffective erythropoiesis. Consistent with the roles of mDia1 and miR-146a in the innate immune response, the serum levels of TNFα and IL-6 were significantly elevated in mDia1/miR-146a double knockout mice. Pathogen-associated molecular pattern proteins (PAMPs), or damage-associated molecular pattern proteins (DAMPs), whose levels increase in aged microenvironment, both induced TNFα and IL-6 upregulation in mDia1/miR-146a double knockout granulocytes and T cells. Mechanistically, we demonstrated that the anemia and ineffective erythropoiesis was independent of hepcidin expression in mDia1/miR-146a double knockout mice. Instead, pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal erythropoiesis through reactive oxygen species-induced caspase-3 activation and cell apoptosis. Our study highlights the dual roles of age-related microenvironment and cytogenetic abnormalities in the pathogenesis of ineffective erythropoiesis in MDS. Disclosures No relevant conflicts of interest to declare.

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Role of Exosomes in Hepcidin Regulation in β-Thalassemia

Blood ◽

10.1182/blood-2019-131094 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 954-954

Author(s):

Marc Ruiz Martinez ◽

Melanie Castro-Mollo ◽

Navneet Dogra ◽

Wenbin An ◽

Ester Borroni ◽

...

Keyword(s):

Bone Marrow ◽

Iron Overload ◽

Iron Metabolism ◽

Wild Type Mouse ◽

Negative Regulator ◽

Mouse Serum ◽

Wild Type ◽

Ineffective Erythropoiesis ◽

Thalassemic Patient ◽

Hepcidin Expression

β-thalassemia is characterized by ineffective erythropoiesis and iron overload. Ineffective erythropoiesis causes iron overload by suppressing hepcidin, the main negative regulator of iron absorption and recycling, and is mediated by secretion of erythroferrone from bone marrow cells. Targeted treatment for ineffective erythropoiesis is unavailable. Furthermore, molecular mechanisms involved in ineffective erythropoiesis and the details of how erythropoiesis regulates iron metabolism are incompletely understood. Lastly, while loss of erythroferrone in β-thalassemic mice leads to partial reversal of iron overload [Kautz Blood 2015], erythroferrone ablated mice are still able to suppress hepcidin after phlebotomy [Kautz Nat Med 2014]. These finding provide evidence of additional regulatory crosstalk between erythropoiesis and iron metabolism. We hypothesize that bone-marrow derived exosomes regulate iron metabolism by modulating hepcidin. Exosomes are small extracellular vesicles derived from multi-vesicular bodies forming intraluminal vesicles which fuse with the plasma membrane and are released by many different cell types [Thery Nat Rev Immun 2002]. In light of their capacity for cell-cell communication and modification of the microenvironment, exosomes have been widely studied in multiple diseases [Valadi Nat Cell Bio 2007] despite which, erythropoiesis-derived exosomes and their role in iron metabolism regulation remain unexplored. Our preliminary data demonstrate that phlebotomy in wild type mice results in increased exosome concentration in serum and that exosomes are increased in th3/+ mouse serum (Figure 1a). Furthermore, hepcidin induction by exosome depleted-FBS is decreased relative to FBS (Figure 1b), and exosomes isolated from FBS induce hepcidin in a dose response manner in vitro (Figure 1c). We thus propose to explore the mechanistic relationship between exosomes and hepcidin regulation in β-thalassemia. Serum samples from patients with β-thalassemia major and age / gender matched controls were collected; all patients were treated with iron chelation therapy and all samples were collected immediately prior to transfusion. Exosome fractions were purified and analyzed in patients relative to controls. Although there is no difference in the number of exosomes or mean particle size within the exosomal fraction, exosomal protein content per volume of serum is significantly decreased in patients relative to controls. In addition, the treatment of primary wild type mouse hepatocytes with sera from patients and controls reveals the expected relatively decreased hepcidin induction in β-thalassemic patient sera treated hepatocytes relative to control sera; a similar difference is seen in hepatocytes treated with exosome-depleted sera from patients and controls (Figure 2a). These findings suggest that hepcidin suppression is a consequence of the exosome-free portion of serum from control and β-thalassemic samples. Furthermore, only exosomes derived from β-thalassemic patient sera induces hepcidin expression in primary wild type mouse hepatocyte cultures (Figure 2b). Lastly, exosomes derived from β-thalassemic patient sera do not affect ERK1/2 and STAT3 signaling in primary hepatocytes but increase SMAD1/5/8 (Figure 2c) and decrease AKT signaling (Figure 2d). Taken together, these findings demonstrate that exosomes enhance hepcidin expression via increased SMAD1/5/8 signaling, that increased hepcidin may influence multiple signaling pathways by an autocrine mechanism in response to exosomes, and that exosomes counterbalance hepcidin suppressive substances in the exosome-depleted serum from β-thalassemic samples. Our studies provide novel insights into the important previously unexplored mechanism of hepcidin regulation by exosomes in both physiologic and pathologic states. Disclosures Coates: apo pharma: Consultancy, Honoraria, Speakers Bureau; vifor: Consultancy, Honoraria; celgene: Consultancy, Honoraria, Other: steering committee of clinical study; agios pharma: Consultancy, Honoraria. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees.

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Apotransferrin Injection Leads to More Effective Erythropoiesis in β-Thalassemic Mice.

Blood ◽

10.1182/blood.v110.11.276.276 ◽

2007 ◽

Vol 110 (11) ◽

pp. 276-276

Author(s):

Yelena Z. Ginzburg ◽

Anne C. Rybicki ◽

Sandra M. Suzuka ◽

Leni von Bonsdorff ◽

Mary E. Fabry ◽

...

Keyword(s):

Bone Marrow ◽

Iron Overload ◽

Binding Capacity ◽

Globin Gene ◽

Transferrin Saturation ◽

Dry Weight ◽

Ineffective Erythropoiesis ◽

Erythroid Precursor ◽

Hepcidin Expression ◽

Erythroid Precursors

Abstract β-thalassemia is a disease resulting from a β-globin gene mutation which leads to less β-globin, expanded and ineffective erythropoiesis, and anemia. Additionally, mature red blood cells have a shortened survival. Although the degree of anemia varies, from severe transfusion-dependence to only an increase in iron absorption in the gut to maintain hemoglobin levels, all thalassemic patients develop some degree of iron overload. In a previous study using β-thalassemic mice, we were able to induce iron overload using iron dextran injections and demonstrated an increase in hemoglobin with increased reticulocytosis and an expansion of extramedullary erythropoiesis in the liver and spleen. The fact that iron administration reduced anemia in β-thalassemic mice was surprising. Since patients with β-thalassemia have ample iron supply, we hypothesized that part of the anemia in β-thalassemia may result from a maldistribution of iron as a consequence of insufficient circulating transferrin to deliver iron for erythropoiesis. In the present study, we analyzed the effect of intraperitoneal human apotransferrin injections on markers of hematopoiesis and iron metabolism in thalassemic mice. We used three different doses of apotransferrin - 5mg, 10mg, and 30mg - daily, for a 10 day course. Mice with β-thalassemia intermedia (Hbbth1/th1) were compared with age and gender matched control C57BL/6J mice. Although no increase in hemoglobin was observed, the reticulocyte count decreased after apotransferrin injections (2975±125 x 109 vs. 1636±130 x 109 cells/L, P=2.29 x 10−5). The efficiency of erythropoiesis, as measured by red cell to reticulocyte ratio, increased after apotransferrin injections (0.029±0.002 vs. 0.007±0.0007, P=0.0002), confirming that more red cells circulate as a result of each maturing erythroid precursor. We were able to demonstrate that apotransferrin is effective in increasing transferrin iron binding capacity (TIBC) (739.5±98.4 vs. 419.1±18.3 mg/dL, P=0.002) without changing the transferrin saturation (23.0±7.1 vs. 34.9±4.2%, P=0.15) in our mice. Apotransferrin injections also resulted in a reduction of iron deposition in the liver (5.49±0.48 vs. 11.08±1.24 mg/g dry weight, P=0.003) and heart (1.25±0.18 vs. 2.26±0.26 mg/g dry weight, P=5.7 x 10−5) of Hbbth1/th1 mice without changes in labile plasma iron levels. Using flow cytometry, we demonstrated an increase in erythroid precursors in the bone marrow of Hbbth1/th1 mice (68.7±1.5 vs. 56.5±3.96% ter119+ precursors, P=0.01) but a decrease in the spleen (41.05±3.16 vs. 60.95±8.3% ter119+ precursors, P=0.03) compared to baseline. Lastly, liver hepcidin expression was progressively suppressed with increasing transferrin dose in our mice. Taken together, this data strongly suggests that exogenous apotransferrin is able to mobilize stored iron for production of erythroid precursors in the bone marrow; this process leads to hepcidin suppression. Diseases of ineffective erythropoiesis, in which expanding erythropoiesis may be limited by the iron delivery system to maintain hemoglobin production, may be a result of insufficient transferrin and relative iron deficiency. The significance of our current findings has potential broad implications for the mobilization of stored iron for use in erythropoiesis in many diseases in which iron overload co-exists with anemia such as β-thalassemia, sideroblastic anemia, and the myelodysplastic syndromes.

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Increased Transferrin Concentration Ameliorates Anemia in Beta-Thalassemic Mice Through Changes in Iron Uptake and TfR1 Trafficking

Blood ◽

10.1182/blood.v118.21.906.906 ◽

2011 ◽

Vol 118 (21) ◽

pp. 906-906

Author(s):

Huihui Li ◽

Lionel Blanc ◽

Tenzin Choesang ◽

Maya Shvartsman ◽

Leni vonBonsdorff ◽

...

Keyword(s):

Cell Culture ◽

Bone Marrow ◽

Plasma Membrane ◽

Iron Uptake ◽

Erythroid Differentiation ◽

Beta Thalassemia ◽

Cellular Iron ◽

Cellular Iron Uptake ◽

Iron Delivery ◽

Terminal Erythroid Differentiation

Abstract Abstract 906 Hemoglobin (Hb) synthesis during terminal erythroid differentiation is iron dependent and iron delivery requires transferrin (Tf) to transferrin receptor 1 (TfR1) binding. After binding of Tf to TfR1, the ligand-receptor complex is endocytosed through a clathrin dependent mechanism, results in iron delivery via the endosomal compartment, and ends with TfR1 recycling back to the plasma membrane. During erythroid differentiation, TfR1 expression is downregulated and is completely absent from the mature red blood cells (RBCs). Reticulocyte TfR1 is rerouted from the endosomal recycling pathway by sorting to exosomes where it is shed from the cell. Exosomes are small membrane vesicles originating from the fusion of a multi-vesicular endosome compartment with the plasma membrane, leading to the secretion of intraluminal vesicles into circulation. Cell surface TfR1 expression is proportional to the concentration of soluble TfR1 found in circulation as a consequence of exosomal secretion and is increased both in iron deficiency and expanded erythropoiesis. Increased soluble TfR1 is observed in disease of expanded and ineffective erythropoiesis (IE) such as beta-thalassemia, a disease associated with anemia, extramedullary hematopoiesis (EMH), and splenomegaly. We previously demonstrated that apoTf-treated beta-thalassemic mice have more circulating RBCs, increased Hb, reversed splenomegaly and EMH, with fewer reticulocytes and erythroid precursors in the bone marrow and spleen. Furthermore, in both beta-thalassemic and C57BL/6 mice treated with apoTf, we observed a significant reduction in mean cellular hemoglobin (MCH). We hypothesize that TfR1 trafficking is impaired in beta-thalassemia and that exogenous apoTf reduces cellular iron uptake and normalizes TfR1 trafficking pathways, resulting in reduced heme synthesis and a lower MCH observed in apoTf-treated mice. To test this hypothesis, we evaluate 1) cellular iron uptake in cell culture and 2) TfR1 mRNA expression, cell surface expression, and endosomal/exosomal trafficking pathways in C57BL/6 and beta-thalassemic mice. Cell culture experiments were performed using K562 cells +/− 50- and 250-fold excess apoTf relative to holoTf. Mice were evaluated after 20 days of 10 mg human apoTf IP injections (compared with PBS injection). Using a calcein fluorescence quenching approach, we demonstrate that exogenous apoTf decreases iron uptake in culture in a dose response manner. Furthermore, in mouse bone marrow samples sorted using CD44/TER119, we show that TfR1 mRNA expression is higher in beta-thalassemic relative to C57BL/6 mice and increases further in apoTf treated mice in all stages of terminal erythroid differentiation. In addition, although western blot experiments show an increase in cellular TfR1 in beta-thalassemic relative to C57BL/6 mice, cell fractionation experiments demonstrate a proportional shift from the plasma membrane to the endosomal compartment in apoTf-treated mice and reticulocytes of apoTf treated beta-thalassemic mice exhibit significantly reduced TfR1 expression per cell. Finally, reticulocyte TfR1 sorting into exosomes is impaired in beta-thalassemic relative to C57BL/6 mice with a proportional increase in exosomal TfR1 clearance and a reduction in soluble TfR1 in the serum in apoTf treated beta-thalassemic mice. Taken together, our findings demonstrate for the first time that TfR1 trafficking is important for RBC physiology separately from its role in iron uptake and that exogenous apoTf enhances TfR1 endosomal trafficking, normalizes TfR1 sorting into exosomes, and reduces cellular iron uptake. Finally, our results elucidate mechanisms by which MCH is reduced in apoTf-treated mice and provide evidence for multiple consequences of Tf:TfR1 binding on erythroid differentiation and proliferation that characterize diseases of IE. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of glypican-1 expression induces an activated fibroblast phenotype in a human bone marrow-derived stromal cell-line

Scientific Reports ◽

10.1038/s41598-021-88519-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sukhneeraj P. Kaur ◽

Arti Verma ◽

Hee. K. Lee ◽

Lillie M. Barnett ◽

Payaningal R. Somanath ◽

...

Keyword(s):

Prostate Cancer ◽

Bone Marrow ◽

Cancer Cells ◽

Cancer Cell ◽

Stromal Cell ◽

Prostate Cancer Cell ◽

Bone Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Prostate Cancer Cells

AbstractCancer-associated fibroblasts (CAFs) are the most abundant stromal cell type in the tumor microenvironment. CAFs orchestrate tumor-stromal interactions, and contribute to cancer cell growth, metastasis, extracellular matrix (ECM) remodeling, angiogenesis, immunomodulation, and chemoresistance. However, CAFs have not been successfully targeted for the treatment of cancer. The current study elucidates the significance of glypican-1 (GPC-1), a heparan sulfate proteoglycan, in regulating the activation of human bone marrow-derived stromal cells (BSCs) of fibroblast lineage (HS-5). GPC-1 inhibition changed HS-5 cellular and nuclear morphology, and increased cell migration and contractility. GPC-1 inhibition also increased pro-inflammatory signaling and CAF marker expression. GPC-1 induced an activated fibroblast phenotype when HS-5 cells were exposed to prostate cancer cell conditioned media (CCM). Further, treatment of human bone-derived prostate cancer cells (PC-3) with CCM from HS-5 cells exhibiting GPC-1 loss increased prostate cancer cell aggressiveness. Finally, GPC-1 was expressed in mouse tibia bone cells and present during bone loss induced by mouse prostate cancer cells in a murine prostate cancer bone model. These data demonstrate that GPC-1 partially regulates the intrinsic and extrinsic phenotype of human BSCs and transformation into activated fibroblasts, identify novel functions of GPC-1, and suggest that GPC-1 expression in BSCs exerts inhibitory paracrine effects on the prostate cancer cells. This supports the hypothesis that GPC-1 may be a novel pharmacological target for developing anti-CAF therapeutics to control cancer.

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CBIO-10. REDUCED IRON EXPORT FUNCTIONS IN A CELL INTRINSIC MANNER TO DRIVE GLIOBLASTOMA GROWTH

Neuro-Oncology ◽

10.1093/neuonc/noaa215.070 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii17-ii17

Author(s):

Katie Troike ◽

Erin Mulkearns-Hubert ◽

Daniel Silver ◽

James Connor ◽

Justin Lathia

Keyword(s):

Tumor Cells ◽

Iron Uptake ◽

Iron Homeostasis ◽

Iron Status ◽

Tumor Grade ◽

Hfe Gene ◽

Iron Release ◽

Female Patients ◽

Cellular Iron

Abstract Glioblastoma (GBM), the most common primary malignant brain tumor in adults, is characterized by invasive growth and poor prognosis. Iron is a critical regulator of many cellular processes, and GBM tumor cells have been shown to modulate expression of iron-associated proteins to enhance iron uptake from the surrounding microenvironment, driving tumor initiation and growth. While iron uptake has been the central focus of previous investigations, additional mechanisms of iron regulation, such as compensatory iron efflux, have not been explored in the context of GBM. The hemochromatosis (HFE) gene encodes a transmembrane glycoprotein that aids in iron homeostasis by limiting cellular iron release, resulting in a sequestration phenotype. We find that HFE is upregulated in GBM tumors compared to non-tumor brain and that expression of HFE increases with tumor grade. Furthermore, HFE mRNA expression is associated with significantly reduced survival specifically in female patients with GBM. Based on these findings, we hypothesize that GBM tumor cells upregulate HFE expression to augment cellular iron loading and drive proliferation, ultimately leading to reduced survival of female patients. To test this hypothesis, we generated Hfe knockdown and overexpressing mouse glioma cell lines. We observed significant alterations in the expression of several iron handling genes with Hfe knockdown or overexpression, suggesting global disruption of iron homeostasis. Additionally, we show that knockdown of Hfe in these cells increases apoptosis and leads to a significant impairment of tumor growth in vivo. These findings support the hypothesis that Hfe is a critical regulator of cellular iron status and contributes to tumor aggression. Future work will include further exploration of the mechanisms that contribute to these phenotypes as well as interactions with the tumor microenvironment. Elucidating the mechanisms by which iron effulx contributes to GBM may inform the development of next-generation targeted therapies.

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