Uptake Of Transferrin-bound Iron Research Articles

Modulation of Duodenal TfR Expression in IDA Induced Rat with Date Palm and Goat Milk Intake

Iron deficiency anemia (IDA) is a medical condition characterized by insufficient iron levels in the body, resulting in a reduced ability to produce hemoglobin (Hb), a critical component of red blood cells. IDA is often associated with chronic fatigue, impaired cognitive function, and diminished well-being. Transferrin (Tf) is one of the major proteins in iron homeostasis, responsible for transporting iron through the blood to various tissues, while its carrier protein, transferrin receptor (TfR), mediates the cellular uptake of transferrin-bound iron into the cell. This study aims to evaluate the expression of TfR mRNA and protein in the small intestine following intervention with date palm and goat milk in IDA-induced rats. Twenty-four male Wistar rats were induced with IDA for 2 weeks using a low-iron diet. Following IDA detection, rats were supplemented with date palm and goat milk, singly and in combination. After four weeks, the rats were sacrificed, and the expression of TfR mRNA and protein in the small intestine was assessed using qPCR and immunohistochemistry, respectively. Data were analyzed using SPSS 24.0, with a significance level set at p<0.05. Results demonstrated that date palm and goat milk significantly improved Hb, serum iron, Tf saturation levels, and modulated the expression of TfR mRNA in the IDA-induced rats. Expression of TfR on the crypt region and brush border membrane of the small intestine was normalized following intervention. The findings indicate that supplementation of date palm and goat milk improved Hb and Tf saturation levels and significantly modulated duodenal TfR expression in IDA-induced rats.

Brain iron acquisition depends on age and sex in iron-deficient mice.

Adequate and timely delivery of iron is essential for brain development. The uptake of transferrin-bound (Tf) iron into the brain peaks at the time of myelination, whereas the recently discovered H-ferritin (FTH1) transport of iron into the brain continues to increase beyond the peak in myelination. Here, we interrogate the impact of dietary iron deficiency (ID) on the uptake of FTH1- and Tf-bound iron. In the present study, we used C57BL/6J male and female mice at a developing (post-natal day (PND) 15) and adult age (PND 85). In developing mice, ID results in increased iron delivery from both FTH1 and Tf for both males and females. The amount of iron uptake from FTH1 was higher than the Tf and this difference between the iron delivery was much greater in females. In contrast, in the adult model, ID was associated with increased brain iron uptake by both FTH1 and Tf but only in the males. There was no increased uptake from either protein in the females. Moreover, transferrin receptor expression on the microvasculature as well as whole brain iron, and H and L ferritin levels revealed the male brains became iron deficient but not the female brains. Last, under normal dietary conditions, 55 Fe uptake was higher in the developing group from both delivery proteins than in the adult group. These results indicate that there are differences in iron acquisition between the developing and adult brain for FTH1 and Tf during nutritional ID and demonstrate a level of regulation of brain iron uptake that is age and sex-dependent.

Macrophage Resistance to Ionizing Radiation Exposure Is Accompanied by Decreased Cathepsin D and Increased Transferrin Receptor 1 Expression.

To identify a molecular signature of macrophages exposed to clinically relevant ionizing radiation (IR) doses, mirroring radiotherapy sessions. Human monocyte-derived macrophages were exposed to 2 Gy/ fraction/ day for 5 days, mimicking one week of cancer patient's radiotherapy. Protein expression profile by proteomics was performed. A gene ontology analysis revealed that radiation-induced protein changes are associated with metabolic alterations, which were further supported by a reduction of both cellular ATP levels and glucose uptake. Most of the radiation-induced deregulated targets exhibited a decreased expression, as was the case of cathepsin D, a lysosomal protease associated with cell death, which was validated by Western blot. We also found that irradiated macrophages exhibited an increased expression of the transferrin receptor 1 (TfR1), which is responsible for the uptake of transferrin-bound iron. TfR1 upregulation was also found in tumor-associated mouse macrophages upon tumor irradiation. In vitro irradiated macrophages also presented a trend for increased divalent metal transporter 1 (DMT1), which transports iron from the endosome to the cytosol, and a significant increase in iron release. Irradiated macrophages present lower ATP levels and glucose uptake, and exhibit decreased cathepsin D expression, while increasing TfR1 expression and altering iron metabolism.

Genome-Wide CRISPR/Cas9-Mediated Loss-of-Function Screening in Human Erythroleukemic Cells Identifies Novel Cellular Components Involved in Iron and Heme Trafficking

To meet the high demand for iron during erythropoiesis, erythroid precursors utilize efficient yet tightly regulated mechanisms of iron trafficking. Transferrin-bound iron (TBI), the major cellular iron source for erythroid cells under physiological conditions, is taken up through the transferrin receptor (TfR) by endocytosis. Non-transferrin bound iron (NTBI), which exists in the circulation under conditions of iron overload, is also utilized by erythroid cells and its uncontrolled uptake by erythroid precursors presumably results in ineffective erythropoiesis (IE). The majority of iron acquired by erythroid cells is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. In this study, we employed a functional approach to identify cellular components involved in TBI and NTBI uptake and heme trafficking in human erythroid cells. We performed genome-wide loss-of-function screens using the CRISPR-Cas9 system in K562 erythroleukemic cells, which can utilize either TBI or NTBI as an essential iron source for proliferation and can be differentiated into erythroid cells by hemin. In addition to genes that are known to be involved in the studied processes, our approach revealed novel candidates whose roles in iron uptake, heme trafficking and erythroid differentiation were validated using targeted confirmatory approaches. Our screen on iron uptake identified 25 genes potentially involved in TBI acquisition in addition to 18 NTBI uptake candidate genes. The transferrin receptor (TfR1) was one of the top TBI uptake candidates indicating that our approach effectively revealed mechanistically relevant components in the process. Unsurprisingly, several components of the endocytic pathway were shown potentially indispensable for transferrin acquisition. We identified a role for the vacuolar-type H+ - ATPase (V-ATPase) assembly factor CCDC115 in TBI uptake and validated this role by generating individual CCDC115 deficient K562 cells. Our study on heme trafficking revealed several candidate genes relevant to cellular heme uptake, heme-induced differentiation and heme export. Pathway analysis indicates a major role for clathrin-mediated endocytosis and vesicle acidification in heme acquisition by K562 cells. Interestingly, we demonstrated that CCDC115, which was also identified as a significant heme uptake candidate, is indispensable for heme-induced hemoglobinization. We further identified components involved in heme detoxification including two ATP-binding cassette (ABC) transporters that could play a critical unrevealed role in mitochondrial and cellular heme export. Our work demonstrates the strength of functional genomics in studying the various mechanisms affecting erythropoiesis. DisclosuresFraenkel:Sanofi: Employment, Other: Sanofi had no role in funding or influencing this study.

Purification of pancreatic endocrine subsets reveals increased iron metabolism in beta-cells

Abstract Objectives The main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. Methods We developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. Results Pancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. Conclusions This study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.

Purification of pancreatic endocrine subsets reveals increased iron metabolism in beta cells

ObjectivesThe main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. MethodsWe developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. ResultsPancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. ConclusionsThis study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.

Genetic screens reveal CCDC115 as a modulator of erythroid iron and heme trafficking.

Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells.

WDR45 Contributes to Iron Accumulation Through Dysregulation of Neuronal Iron Homeostasis

Abstract Objectives Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by an abnormal accumulation of brain iron and progressive degeneration of the nervous system. β-propeller protein-associated neurodegeneration (BPAN) (OMIM #300,894) is a recently identified subtype of NBIA. BPAN is caused by de novo mutations in the WD repeat domain 45 (WDR45) gene. WDR45 deficiency in BPAN patients and animal models has shown defects in autophagic flux, suggesting a role for WDR45 in autophagy. How WDR45 deficiency leads to brain iron overload remains unclear. The goal of the present study is to identify the pathogenic mechanisms of WDR45 deficiency that cause iron overload and neurodegeneration. Methods To elucidate the role of WDR45 in dopaminergic neuronal cells, we generated a WDR45-knockout (KO) SH-SY5Y cell line by CRISPR/Cas9-mediated genome editing. To identify mechanisms underlying iron homeostasis and transport, we examined two cellular iron acquisition pathways in these cells using radioactive isotope 59Fe: 1) the canonical transferrin-bound iron (TBI) uptake pathway and 2) the nontransferrin-bound iron (NTBI) pathway. Results Loss of WDR45 increased total iron levels with a concomitant increase in the iron storage protein ferritin in neuronal cells. Specifically, WDR45-KO cells preferentially took up NTBI compared to wild-type cells. Concordant with these functional data, the level of divalent metal transporter-1 (DMT1) expression was upregulated in WDR45-KO cells, providing a causal link to iron overload in WDR45 deficiency. In addition, loss of WDR45 led to defects in autophagic flux and impaired ferritinophagy, a lysosomal process that promotes ferritin degradation, suggesting that iron overload is driven by impaired ferritinophagy. Interestingly, WDR45 deficiency increased iron accumulation in the mitochondria, impaired mitochondrial function, and in turn, elevated reactive oxygen species generation. Conclusions Our study provides the first evidence that WDR45 deficiency alters cellular iron acquisition pathways thereby leading to iron accumulation in neuronal cells. These findings will serve as a basis for developing therapeutic strategies for patients with NBIA. Funding Sources NIH, NBIA Disorder Association.

Ascorbate and Tumor Cell Iron Metabolism: The Evolving Story and Its Link to Pathology.

Significance: Vitamin C or ascorbate (Asc) is a water-soluble vitamin and an antioxidant that is involved in many crucial biological functions. Asc's ability to reduce metals makes it an essential enzyme cofactor. Recent Advances: The ability of Asc to act as a reductant also plays an important part in its overall role in iron metabolism, where Asc induces both nontransferrin-bound iron and transferrin-bound iron uptake at physiological concentrations (∼50 μM). Moreover, Asc has emerged to play an important role in multiple diseases and its effects at pharmacological doses could be important for their treatment. Critical Issues: Asc's role as a regulator of cellular iron metabolism, along with its cytotoxic effects and different roles at pharmacological concentrations, makes it a candidate as an anticancer agent. Ever since the controversy regarding the studies from the Mayo Clinic was finally explained, there has been a renewed interest in using Asc as a therapeutic approach toward cancer due to its minimal side effects. Numerous studies have been able to demonstrate the anticancer activity of Asc through selective oxidative stress toward cancer cells via H2O2 generation at pharmacological concentrations. Studies have demonstrated that Asc's cytotoxic mechanism at concentrations (>1 mM) has been associated with decreased cellular iron uptake. Future Directions: Recent studies have also suggested other mechanisms, such as Asc's effects on autophagy, polyamine metabolism, and the cell cycle. Clearly, more has yet to be discovered about Asc's mechanism of action to facilitate safe and effective treatment options for cancer and other diseases.

MiR-148a regulates expression of the transferrin receptor 1 in hepatocellular carcinoma

Transferrin receptor 1 (TFR1) is a transmembrane glycoprotein that allows for transferrin-bound iron uptake in mammalian cells. It is overexpressed in various cancers to satisfy the high iron demand of fast proliferating cells. Here we show that in hepatocellular carcinoma (HCC) TFR1 expression is regulated by miR-148a. Within the TFR1 3′UTR we identified and experimentally validated two evolutionarily conserved miRNA response elements (MREs) for miR-148/152 family members, including miR-148a. Interestingly, analyses of RNA sequencing data from patients with liver hepatocellular carcinoma (LIHC) revealed a significant inverse correlation of TFR1 mRNA levels and miR-148a. In addition, TFR1 mRNA levels were significantly increased in the tumor compared to matched normal healthy tissue, while miR-148a levels are decreased. Functional analysis demonstrated post-transcriptional regulation of TFR1 by miR-148a in HCC cells as well as decreased HCC cell proliferation upon either miR-148a overexpression or TFR1 knockdown. We hypothesize that decreased expression of miR-148a in HCC may elevate transferrin-bound iron uptake, increasing cellular iron levels and cell proliferation.

(Z)-ligustilide increases ferroportin1 expression and ferritin content in ischemic SH-SY5Y cells

The mechanisms involved in the antioxidant and anti-apoptotic properties of (Z)-ligustilide (LIG) are not fully elucidated. Based on the accumulated data, we hypothesized that LIG might be able to reduce ischemia/reperfusion-induced increase in brain iron by regulating expression of iron transport proteins. We therefore investigated the effects of LIG on iron uptake protein transferrin receptor 1, iron exporter protein ferroportin 1, iron storage protein ferritin light chain and also hypoxia inducible factor-1 alpha (HIF-1 alpha) in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated SH-SY5Y cells, using Western blot analysis. We demonstrated that LIG completely reversed the OGD/R-induced reduction of ferroportin 1, increased ferritin light chain content, and also suppressed the OGD-induced increase in HIF-1 alpha in SH-SY5Y cells. These findings imply that LIG might reduce the OGD/R-induced increase in brain iron by promoting cell iron release and iron corporation into ferritin, and also by inhibiting the HIF-1 alpha-induced increase in transferrin-bound iron uptake and iron accumulation in the brain, consequently attenuating iron-mediated free radical formation, oxidative stress and apoptosis.

Cell-permeable iron inhibits vascular endothelial growth factor receptor-2 signaling and tumor angiogenesis.

Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis in vivo. In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis.

Alpha-synuclein modulates retinal iron homeostasis by facilitating the uptake of transferrin-bound iron: Implications for visual manifestations of Parkinson's disease

Aggregation of α-synuclein (α-syn) in neurons of the substantia nigra is diagnostic of Parkinson's disease (PD), a neuro-motor disorder with prominent visual symptoms. Here, we demonstrate that α-syn, the principal protein involved in the pathogenesis of PD, is expressed widely in the neuroretina, and facilitates the uptake of transferrin-bound iron (Tf-Fe) by retinal pigment epithelial (RPE) cells that form the outer blood-retinal barrier. Absence of α-syn in knock-out mice (α-syn−/−) resulted in down-regulation of ferritin in the neuroretina, indicating depletion of cellular iron stores. A similar phenotype of iron deficiency was observed in the spleen, femur, and brain tissue of α-syn−/− mice, organs that utilize mainly Tf-Fe for their metabolic needs. The liver and kidney, organs that take up significant amounts of non-Tf-bound iron (NTBI), showed minimal change. Evaluation of the underlying mechanism in the human RPE47 cell line suggested a prominent role of α-syn in the uptake of Tf-Fe by modulating the endocytosis and recycling of transferrin (Tf)/transferrin-receptor (TfR) complex. Down-regulation of α-syn in RPE cells by RNAi resulted in the accumulation of Tf/TfR complex in common recycling endosomes (CREs), indicating disruption of recycling to the plasma membrane. Over-expression of exogenous α-syn in RPE cells, on the other hand, up-regulated ferritin and TfR expression. Interestingly, exposure to exogenous iron increased membrane association and co-localization of α-syn with TfR, supporting its role in iron uptake by the Tf/TfR complex. Together with our observations indicating basolateral expression of α-syn and TfR on RPE cells in vivo, this study reveals a novel function of α-syn in the uptake of Tf-Fe by the neuroretina. It is likely that retinal iron dyshomeostasis due to impaired or altered function of α-syn contributes to the visual symptoms associated with PD.

Abstract 14830: Cellular Iron Transport Protein, Transferrin Receptor 1 Plays an Important Role in the Pathophysiology of Chronic Kidney Disease

Background: Cellular iron transport protein, transferrin receptor 1 (TfR1) is required for the uptake of transferrin-bound iron into the cells. Several reports have shown that iron accumulation is associated with the pathophysiology of chronic kidney disease (CKD); however, the role of TfR1 in the pathophysiology of CKD remains unknown. Herein, we investigate TfR1 in the experimental models of CKD. Methods and Results: CKD was induced by 5/6 nephrectomy in Sprague-Dawley rats. CKD rats showed proteinuria and tubular dilatation. Of note, TfR1 expression is increased in the remnant kidney along with increased iron accumulation, as compared with controls. To investigate the functional importance of TfR1 in the pathophysiology of CKD, we subjected to 5/6 nephrectomy in TfR1 hetero knockout mice. At 8weeks after 5/6 nephrectomy, systolic blood pressure was similar in TfR1 hetero knockout mice and wild-type (WT) littermates. In contrast, urinary albumin excretion, serum BUN, and creatinine levels were increased to a lesser extent in TfR1 hetero knockout mice compared with WT littermates. In addition, renal fibrosis was observed in the remnant kidney in WT mice, while the extent was attenuated in TfR1 hetero knockout mice. Consistent with these findings, increased renal gene expression of collagen type I and collagen type III were attenuated in TfR1 hetero knockout mice compared with WT littermates. Conclusions: These results indicate that TfR1 plays a role in the pathophysiology of CKD. Understanding the role of TfR1 in the pathophysiology of CKD may lead to a novel therapeutic approach for CKD.

Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology.

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail.

Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology.

Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera.

Abstract 12816: Cellular Iron Transport Protein, Transferrin Receptor 1 Plays a Role in the Pathophysiology of Pulmonary Arterial Hypertension

Introduction: Cellular iron transport protein, transferrin receptor 1 (TfR1) is required for the uptake of transferrin-bound iron into the cells. Some reports have shown that iron deficiency is reported to be prevalent in patients with pulmonary arterial hypertension (PAH); however, the role of TfR1 in the pathophysiology of PAH remains unknown. Hypothesis: We hypothesiged that TfR1 plays a role in the pathophysiology of PAH. Here, we investigate TfR1 in the experimental models of PAH. Methods: Male Sprague-Dawley rats were subcutaneously injected with monocrotaline (60 mg/kg). Saline-injected rats were served as controls. In addition, we knocked down TfR1 expression in human pulmonary arterial smooth muscle cells (hPASMCs) and primary cardiomyocytes prepared from neonatal rats using its siRNA. Results: Monocrotaline-injected rats showed pulmonary vascular remodeling, increased right ventricular pressure, and right ventricular hypertrophy. Of interest, TfR1 expression was increased in the pulmonary remodeled artery and the hypertrophied right ventricle of monocrotaline-injected rats, as compared with the controls. Meanwhile, iron content did not differ in the pulmonary artery and right ventricle between the control and monocrotaline-injected rats. Transfection of hPASMCs and cardiomyocytes with TfR1 siRNA significantly reduced TfR1 gene expression. Importantly, TfR1 silencing suppressed platelet-derived growth factor (PDGF)-induced proliferation of hPASMCs. Moreever, TfR1 silencing attenuated phenylephrine-induced increase in cell size of cardiomyocytes. Conclusions: These results indicate that TfR1 plays a role in the pathophysiology of pulmonary artery remodeling and right ventricular hypertrophy in PAH.

Angiotensin II inhibits uptake of transferrin-bound iron but not non-transferrin-bound iron by cultured astrocytes

The existence of all components of the renin–angiotensin system (RAS) and the iron metabolism system, and the recent findings on the functions of angiotensin II (ANGII) in peripheral iron metabolism imply that ANGII might play a role in iron homeostasis by regulating expression of iron transport proteins in the brain. Here, we investigated effects of ANGII on uptake and release of iron as well as expression of cell iron transport proteins in cultured astrocytes. We demonstrated that ANGII could significantly inhibit transferrin-bound iron (Tf-Fe) uptake and iron release as well as the expression of transferrin receptor 1 (TfR1) and the iron exporter ferroportin 1 (Fpn1) in cultured astrocytes. This indicated that the inhibitory role of ANGII on Tf-Fe uptake and iron release is mediated by its negative effect on the expression of TfR1 and Fpn1. We also provided evidence that ANGII had no effect on divalent metal transporter 1 (DMT1) expression as well as non-transferrin-bound iron (NTBI) uptake in the cells. Our findings showed that ANGII has a role to affect expression of iron transport proteins in astrocytes in vitro and also suggested that ANGII might have a physiological function in brain iron homeostasis.

Mutation Of The Divalent Metal Transporter (Dmt1) Gene Results In Inefficient Induction Of The Erythroid Transcriptional Program Due To Latter Onset Of GATA-1 and Epor Expression

IntroductionDivalent metal transporter 1 (DMT1; SLC11A2) encodes trans-membrane protein variants that execute either non-heme iron absorption through apical duodenal membrane of enterocytes or transferrin-bound iron uptake through endosomal membrane of erythroid (and other) cells. Since 2005 several DMT1 mutations affecting accurate protein folding and localization were identified in patients with defective iron uptake leading to microcytic anemia, abnormal growth of erythroid progenitors in vitro, and iron overload in the liver. We have previously demonstrated that defective growth of DMT-1-mutatnt BFU-Es in vitro and anemia associated with ineffective erythropoiesis in vivo can be improved with high-dose erythropoietin (EPO) supplementation (Horvathova et al., 2012). Data from Dmt1-mutant mk/mk mice (Gunshin et al., 2005) suggested that the anemic phenotype is a result of ineffective erythropoiesis within bone marrow and spleen. HypothesisDMT1-mutant erythropoiesis inhibits EPO receptor (EPOR) signaling whose end-point target as well as upstream activator is the key transcription factor GATA-1. This results in defective erythroid development characterized by impaired survival capacity of erythroid progenitors, increased apoptosis of erythroblasts, and dysregulation of erythroid gene expression leading to ineffective erythropoiesis. MaterialsBone marrow cells of murine mk/mk mice were sorted using flow cytometry to obtain differentiating erythroid fractions based on antigens Cd71 and Ter119 (Sokolovsky et al., 2001) which were then subjected to gene expression analysis. ResultsFirstly, four consecutive fractions (proerythroblasts and early basophilic erythroblasts - stage I, late basophilic erythroblasts – stage II, chromathophillic and orthochromathophillic erythroblasts – stage III, and late chromathophillic erythroblasts and reticulocytes – stage IV) were isolated from mk/mk mice and control littermates. Flow cytometry showed enrichment in stages I and II and depletion in stage IV in the mk/mk bone marrow when compared to wild type controls, consistent with our previous data. In the spleen the major cellular enrichment was seen in stage III accompanied by cell depletion in stage IV. Gene expression of GATA-1 was markedly decreased at the onset (in stages I and II) of erythropoiesis while it was increased in terminal stage IV. The expression pattern of the GATA-1 target gene Epor was similar to that of GATA-1 expression, while expression of b-maj globin was significantly reduced indicating developmental delay of the erythroid Dmt1-mutant compartment (compared to wild type). Whereas GATA-1 and Epor expression is low in early erythropoiesis, a compensatory increase in their expression at later stages is not capable to efficiently upregulate b-maj globin. These data together with flow cytometry analysis identify a developmental blockade of erythropoiesis between stages II(III) and IV. To better understand whether Dmt1 levels regulate GATA-1 and Epor expression we used murine erythroleukemia (MEL) cells containing conditional (estrogen-regulated) transgene encoding GATA-1 fused with estrogen receptor ligand-binding domain (GER). MEL cells are cytologically characterized as proerythroblasts and early basophilic erythroblasts, stage I. Firstly, we established that GATA-1 upregulates Epor expression and directly binds to the Epor gene using RT-PCR, Immunoblotting, and chromatin immunoprecipitation (ChIP) in activated GER cells. Using ChIP-sequencing analysis of GATA-1 (and a panel of histone modifications) the GATA-1 enrichment was clearly identified at three distinct Epor regions in murine erythroblasts and differentiating MEL cells. Next, we downregulated Dmt1 using siRNA and observed that GATA-1-mediated upregulation of Epor in activated GER cells became inhibited. In addition, the knockdown of Dmt1 also inhibited steady state levels of GATA-1 in MEL cells by 25%. ConclusionsIneffective erythropoiesis in Dmt1-mutant mice is blocked at stages II (III) and display deregulation of the Epor signaling cascade involving GATA-1 and its targets. Our data thus interconnect iron uptake and the Epor/GATA-1 pathways and suggest their roles during erythroid pathogenesis upon DMT1 mutations. Grants: P305/11/1745, P301/12/P380, P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, GAUK 251135 82210 Disclosures:No relevant conflicts of interest to declare.

Iron Metabolism In Macrophages In Non Transfusion Dependent Thalassemia

Background β-thalassemia is a disease characterized by alteration of β-globin chains production. The phenotype is associated with development of anemia, ineffective erythropoiesis (IE), and iron overload. Cellular iron homeostasis in macrophages is regulated at multiple steps and by numerous genes. Macrophages can acquire iron by Transferrin receptor 1-mediated uptake of transferrin-bound iron, acquisition of molecular iron via the divalent metal transporter 1 (Dmt1) and phagocytosis of senescent erythrocytes with subsequent recycling of iron. There is only one well-characterized pathway by which iron can exit cells, ferroportin-1 (Fpn1), which is expressed on the cell surface of macrophages and acts as the exclusive trans-membrane export protein for ferrous (Fe2+) iron. Hepcidin, a mainly liver-derived peptide induced by iron and cytokines and master regulator of body iron homeostasis, exerts its regulatory effects via binding to Fpn1, which is thought to be the hepcidin receptor. This interaction results in Fpn1 internalization, proteasomal degradation and blockage of iron export. Little is known about what happens at the level of macrophages in thalassemic patients and how they face the high iron concentration. The aim of this study was to characterize the differential expression of the genes involved in iron homeostasis and changes in iron trafficking in fully differentiated unpolarized (M0) human macrophages in Non Transfusion Dependent Thalassemia (NTDT) patients. Methods Monocytes were purified using positive selection with CD14-coated magnetic beads (Miltenyi Biotec) from peripheral blood of 7 NTDT patients and 7 healthy normal controls. Monocytes were cultured for 6 days in RPMI containing 10% FBS and 25 ng/ml GM-CSF and differentiated in mature macrophages. The expression of specific macrophages surface proteins was analyzed by flow cytometry. We used immunohistochemistry to evaluate differentiation and iron retention of macrophages. For basic morphology characterization, formalin-fixed cells were stained using hematoxylin and eosin staining. Iron was detected with DAB-enhanced Perls' staining (Prussian blue reaction). Total mRNA was extracted from cultured cells and gene expression profile was analyzed by real-time PCR using Taqman-probes technologies. Results The purity of the resulting cells suspension was tested by fluorescent-activated cell sorting analysis and was beyond 98%. The cell morphology of macrophages showed no differences between control and thalassemic patients. Using immunohistochemistry iron was not detected in human cultured macrophages of thalassemia patients and controls. We characterized the expression of genes related to iron homeostasis. We analyzed the gene expression levels of SLC40A1 (Ferroportin) , SLC11A2 (Dmt1), HAMP (Hepcidin) that are directly involved in macrophage iron traffic and these results show no statistical differences between patients and controls. Conclusions Due to the heterogeneity of the cellular morphology of macrophages there was no significant difference between the macrophages morphology from NTDT and controls. Iron was not present in M0 magrophages. The gene expression levels of ferroportin, Dmt1 and Hepcidin in mature macrophages grown in regular culture medium without other stimuli were comparable between controls and patients. This culture system does not reflect the in vivo iron metabolism of thalassemic patients due to possibly monocytes inability to internalize and accumulate iron. Different stimuli are necessary to simulate the in vivo condition to differentiate the macrophages. Cells of the monocyte-macrophage lineage are characterized by marked phenotypical and functional heterogeneity. Classical activation by microbial agents and/or Th1 cytokines is associated with the production of oxygen radicals and the pro-inflammatory cytokines involved in cytotoxicity and microbial killing (M1 polarization), but macrophages can also follow a different activation pathway after stimulation with the Th2 cytokines IL-4 or IL-13 (M2 polarization). We will therefore investigate whether iron homeostasis is regulated differently in M1 and M2 macrophages and possibly provide a better understanding of the changes in iron metabolism that take place under thalassemia condition. Disclosures: No relevant conflicts of interest to declare.