Transferrin receptor 2 is frequently expressed in human cancer cell lines

Different proteins ensure the fine control of iron metabolism at the level of various tissues. Among these proteins, it was discovered a second transferrin receptor (TfR2), that seems to play a key role in the regulation of iron homeostasis. Its mutations are responsible for type 3 hemochromatosis (Type 3 HH). Although TfR2 expression in normal tissues was restricted at the level of liver and intestine, we observed that TfR2 was frequently expressed in tumor cell lines. Particularly frequent was its expression in ovarian cancer, colon cancer and glioblastoma cell lines; less frequent was its expression in leukemic and melanoma cell lines. Interestingly, in these tumor cell lines, TfR2 expression was inversely related to that of receptor 1 for transferrin (TfR1). Experiments of in vitro iron loading or iron deprivation provided evidence that TfR2 is modulated in cancer cell lines according to cellular iron levels following two different mechanisms: (i) in some cells, iron loading caused a downmodulation of total TfR2 levels; (ii) in other cell types, iron loading caused a downmodulation of membrane-bound TfR2, without affecting the levels of total cellular TfR2 content. Iron deprivation caused in both conditions an opposite effect compared to iron loading. These observations suggest that TfR2 expression may be altered in human cancers and warrant further studies in primary tumors. Furthermore, our studies indicate that, at least in tumor cells, TfR2 expression is modulated by iron through different biochemical mechanisms, whose molecular basis remains to be determined.     

Regulation of iron absorption and storage iron turnover

The regulation of iron supply to plasma was studied in male rate. Repeated exchange transfusions were first carried out with plasma from iron-deficient or iron-loaded animals. There was no recognizable effect on the amount of iron entering the plasma as evidenced by plasma iron concentration or iron absorption by recipient animals. In other studies, iron compounds having different tissue distribution were injected. Subsequent iron release was greater from reticuloendothelial cells than from other iron-loaded tissues. When requirements for transferrin iron were increased by exchange transfusion with high reticulocyte blood, within minutes there was a doubling of the rate of tissue iron donation. It was concluded from these studies that (1) iron turnover in the plasma is primarily determined by the number of tissue receptors for iron, particularly those of the erythron, (2) that the amount of iron supplied by each donor tissue is dependent on the output of other donor tissues, and (3) that a humoral mechanism regulating iron exchange is unlikely in view of the speed of response and magnitude of changes in plasma iron turnover. It is proposed that there is some direct mechanism that determines the movement of iron from donor tissues to unsaturated transferrin binding sites.     

Effect of transfused reticulocytes on iron exchange

A animal model was developed whereby reticulocyte-rich blood was introduced into normal rats by exchange transfusion. Measurements of plasma iron turnover was made at similar plasma iron concentrations before and after exchange transfusions. High reticulocyte blood obtained from animals rendered iron deficient by diet or by treatment with phenylhydrazine resulted in a mean increase of 86% in internal iron exchange, while the plasma iron turnover was unaffected by exchange with normal red cells. Since iron input from reticuloendothelial cells could have increased due to breakdown of transfused cells, iron absorption was also measured. Within 1 hr and for a least 6 hr after exchange with high reticulocyte blood, mean absorption in six groups of animals was increased over control animals by 50%-130%. The increased plasma iron turnover and absorption was not mediated by a decrease in plasma iron or an increase in unsaturated iron-binding capacity. Indeed, a higher plasma iron and transferrin saturation augmented the movement of iron into the plasma from iron- donating tissues. It is proposed that the donation of iron by transferrin in some way immediately facilitates the procurement of more iron by transferrin.     

Does hepcidin affect erythropoiesis in hemodialysis patients?

INTRODUCTION: Prohepcidin is the precursor of hepcidin, a liver-derived peptide involved in iron metabolism by blocking its intestinal absorption and its release by the reticuloendothelial system. Iron overload and inflammation increase hepcidin expression, whereas anemia and hypoxia suppress it. In the present study prohepcidin levels were determined in the serum of hemodialysis (HD) patients and its correlations with iron metabolism markers, C-reactive protein (CRP) and hematocrit (Hct) were assessed. PATIENTS AND METHODS: Forty-sixHD patients and 22 healthy volunteers were enrolled in the study. Hct, serum prohepcidin, CRP, iron, ferritin, transferrin saturation and transferrin receptors were measured. The weekly erythropoietin dose, last-month intravenous iron dose and the patients' demographics were recorded. RESULTS: In comparison to the healthy volunteers, the HD patients had higher serum ferritin, transferrin receptors and CRP, lower serum iron and similar transferrin saturation and prohepcidin levels. In the patient group prohepcidin levels were negatively correlated with Hct but not with any other of the examined parameters. Multiple linear regression analysis considering age, inflammation, iron adequacy, erythropoietin dose and prohepcidin levels revealed that prohepcidin was the predominant determinant of Hct. CONCLUSIONS: Taking into account the low Hct levels in the HD patients of our study, it seems plausible that the prohepcidin levels assessed in this group are inappropriately high. These functionally high prohepcidin levels may be associated with the factors that inhibit erythropoiesis in HD patients. On the other hand, the absence of other expected correlations indicates that further studies are needed in order to definitely clarify this aspect.     

The orchestration of body iron intake: how and where do enterocytes receive their cues?

Our understanding of how iron transverses the intestinal epithelium has improved greatly in recent years, although the mechanism by which body iron demands regulate this process remains poorly understood. By critically examining the earlier literature in this field and considering it in combination with recent advances we have formulated a model explaining how iron absorption could be regulated by body iron requirements. In particular, this analysis suggests that signals to alter absorption exert a direct effect on mature enterocytes rather than influencing the intestinal crypt cells. We propose that the liver plays a central role in the maintenance of iron homeostasis by regulating the expression of hepcidin in response to changes in the ratio of diferric transferrin in the circulation to the level of transferrin receptor 1. Such changes are detected by transferrin receptor 2 and the HFE/transferrin receptor 1 complex. Circulating hepcidin then directly influences the expression of Ireg1 in the mature villus enterocytes of the duodenum, thereby regulating iron absorption in response to body iron requirements. In this manner, the body can rapidly and appropriately respond to changes in iron demands by adjusting the release of iron from the duodenal enterocytes and, possibly, the macrophages of the reticuloendothelial system. This model can explain the regulation of iron absorption under normal conditions and also the inappropriate absorption seen in pathological states such as hemochromatosis and thalassemia.     

Iron absorption by hypotransferrinaemic mice

Iron absorption rates by homozygous and wild-type mice from a hypotransferrinaemic mouse colony were examined with in vivo tied-off duodenal segments and in vitro incubated duodenal fragments. Enhanced initial rates of mucosal uptake and carcass transfer by homozygotes, compared to wild-types, were observed. The changes in vivo and in in vitro uptake kinetics resemble changes seen in iron deficient or hypoxic mice, suggesting that the liver iron loading shown by homozygotes is due to a failure of the normal mechanism for regulation of iron absorption. In vivo mucosal uptake and carcass transfer of radioiron showed an inverse correlation with liver non-haem iron content in homozygous hypotransferrinaemic mice, suggesting that some degree of control of absorption, albeit at markedly reduced sensitivity, can operate in these mice. No correlation between haemoglobin level and iron absorption was observed in homozygous hypotransferrinaemic mice, suggesting that this regulator of iron absorption does not function in these mice. The precise pathogenic mechanism of the enhanced iron absorption in hypotransferrinaemia remains to be determined. Mucosal transferrin levels were found to parallel serum transferrin levels in homozygotes, heterozygotes and wild-type mice. This supports previous suggestions that mucosal transferrin is derived from plasma transferrin and that the enhancement of iron absorption, by physiological mechanisms, does not require the presence of mucosal transferrin.     

Bioavailability and the mechanisms of intestinal absorption of iron from ferrous ascorbate and ferric polymaltose in experimental animals

The comparative bioavailability from matching quantities of iron in the form of ferrous ascorbate or ferric polymaltose was defined in rats. Studies were carried out in the intact animals under basal conditions and also when requirements for this metal were either increased or decreased by manipulating stores or erythropoietic activity. No significant difference was found in the total quantity of iron absorbed from either salt or complex under any of these circumstances, suggesting that the mucosal mechanism regulating the overall process was common to both. However, the rate of transfer from the lumen into portal blood was distinctive, reaching a maximum with salt at 30 min compared to 24 h for the complex. To explore the possibility that iron from the two sources was initially handled by different subcellular pathways, the radiolabeled compounds were instilled into loops of bowel that had been isolated between ligatures in vivo. Enterocytes were harvested and fractionated, and incorporation into ferritin and transferrin was determined using RIA. From salt, iron appeared rapidly in duodenal but not ileal ferritin, whereas mucosal transferrin increased under conditions of stimulated absorption, suggesting that this protein may act as a shuttle for the metal. In contrast, iron from polymaltose showed a cumulative incorporation into duodenal ferritin over time that correlated with iron absorption, defined by the appearance of radiolabel in the serum and in the carcass; a similar pattern was demonstrable in ileal mucosal cells. Conversely, binding of iron to transferrin was minimal. No iron polymaltose was found within the mucosal cells. It is suggested that the low rate of iron transfer from this ferric complex may reflect its extracellular breakdown in the lumen of the gastrointestinal tract.     

Hand strike-induced hemolysis and adaptations in iron metabolism in Basque ball players

INTRODUCTION: Basque ball players (BBPs) make repeated hand strikes to the ball which involves continuous mechanical trauma. OBJECTIVE: The aim of this work is to describe the hematological variations and changes in iron metabolism occurring in BBPs as a result of acute and continuous practice of this special sport native to Northern Spain. METHODS: 40 healthy male subjects volunteered to participate in this study: 11 subjects who exercise moderately (control group) but do not play Basque ball sport and 29 professional BBPs were studied in two situations: before a match (BM group) and after a match (AM group). The following hematological parameters were determined: red blood cell count (RBC), hemoglobin and hematocrit; MCV, MCH and MCHC. The following variables were measured in serum: iron, ferritin, transferrin, transferrin saturation, proteins, and lactate dehydrogenase (LDH). Proteins and hemoglobin were determined in urine. RESULTS: The BM group showed lower hematocrit, MCV, hemoglobin and serum transferrin levels, and higher LDH than controls. The AM group showed higher RBC, hemoglobin, serum proteins, iron, transferrin and LDH levels, lower plasma volume, and higher urine hemoglobin and protein levels than the BM group. CONCLUSIONS: Basque ball playing induces hemolysis and increases the plasma capability to quench free iron, but its clinical consequences on iron metabolism do not seem to be enough to take an iron intake proportional to the caloric intake. In order to prevent, in the long term, the development of anemia in these peculiar sportsmen, it would be useful that medical teams observe urine blood losses and oxidative stress in these BBPs.     

Iron metabolism in the hemoglobin-deficit mouse: correlation of diferric transferrin with hepcidin expression

The iron requirements of the erythroid compartment modulate the expression of hepcidin in the liver, which in turn alters intestinal iron absorption and iron release from the reticuloendothelial system. We have taken advantage of an inherited anemia of the mouse (hemoglobin deficit, or hbd) to gain insights into the factors regulating hepcidin expression. hbd mice showed a significant anemia but, surprisingly, their iron absorption was not increased as it was in wild-type animals made anemic to a similar degree by dietary iron depletion. In wild-type mice hepatic hepcidin levels were decreased but in hbd animals a significant and unexpected increase was observed. The level of absorption was appropriate for the expression of hepcidin in each case, but in hbd mice did not reflect the degree of anemia. However, this apparent inappropriate regulation of hepcidin correlated with increased transferrin saturation and levels of diferric transferrin in the plasma, which in turn resulted from the reduced capacity of hbd animals to effectively use transferrin-bound iron. These data strengthen the proposal that diferric transferrin is a key indicator of body iron requirements.     

Does plasma transferrin regulate iron absorption?

It has been suggested that transferrin that has recently donated its iron to receptor sites is 'activated' to take up iron more avidly from donor tissues. The hypothesis was tested in vitro in a system in which use was made of the different electrophoretic mobilities of normal and desialated transferrin. Recently desaturated transferrin and native apotransferrin were added in equal amounts to a solution of radioactive ferric citrate to produce various end saturations. The resultant mixture was electrophoresed on 5.4% polyacrilamide gel, which was then sliced and counted for 59Fe counts. The size of the 2 radioactive peaks was then compared and expressed as a ratio. Using this in vitro system no supporting evidence could be found for the hypothesis that diferric transferrin which has just donated its iron is able to bind available iron more avidly than native apotransferrin.     

Cellular iron processing

Iron is transported in the blood plasma, mainly bound to transferrin, but in abnormal conditions other iron containing compounds may become important. These include ferritin, haemopexin-haem, haptoglobin-haemoglobin and non-specific non-transferrin-bound iron, all of which are taken up from the circulation by the liver. Transferrin-bound iron can be used by all types of cells in amounts that depend on their complement of transferrin receptors. Immature erythroid cells are the most active in this function. Investigations using reticulocytes as an example of erythroid cells have demonstrated the presence of two mechanisms for the uptake of ferrous iron. One, a high affinity process disappears as reticulocytes mature. It probably represents the mechanism by which iron derived from transferrin is transported into the cytosol after receptor-mediated endocytosis of the iron-transferrin complex. The other mechanism has a lower affinity for iron, is retained when reticulocytes mature and is probably associated with Na⁺ transport across the cell membrane. The physiological characteristics of the two iron transport processes and the evidence for the above conclusions are summarized in the present paper.     

Evidence for heterogeneity in hereditary hemochromatosis: Evaluation of 174 persons in nine families

Hereditary hemochromatosis is an autosomal recessive disease in which the gene is linked to the HLA system. Investigation of nine unrelated probands and their family members has revealed distinct groups based on biochemical and clinical manifestations of the disease. Four different types of disease expression were identified: Group I—classic hereditary hemochromatosis with elevated transferrin saturation, serum ferritin levels, and liver iron content; Group II—severe iron overload, accelerated disease manifesting at an early age; Group III—elevated total body iron stores, normal transferrin saturation and serum ferritin levels; Group IV—markedly elevated findings on serum biochemical tests, e.g., transferrin saturation, serum ferritin levels, with minimal elevation in total body iron stores. This evidence for several clearly distinguishable modes of expression in different families suggests that more than one genetic lesion in iron metabolism may be responsible for iron overload in hereditary hemochromatosis. This genetic heterogeneity may be helpful in delineating the fundamental abnormalities in iron metabolism in this group of disorders.     

Can iron chelators influence the progression of atherosclerosis?

Epidemiological studies and experimental data suggest iron involvement in atherosclerosis. The relation between iron and atherosclerosis is complex and remains contradictory. In thalassemia patients, non transferrin bound iron (NTBI) and free hemoglobin (Hb) are present in plasma and may accelerate atherogenesis, but its progression may be inhibited by iron chelators. The mechanism whereby iron may stimulate atherogenesis has been intensively investigated. Non transferrin bound iron and sera from subjects with hemochromatosis induced endothelial activation with expression of vascular adhesion molecules and endothelial inflammatory chemokines. Such events could be inhibited by iron chelators and oxygen radical scavengers with intracellular activity. Iron chelators may be effective in preventing vascular damage in patients with high concentrations of NTBI as found in thalassemia.     

Non-Transferrin Plasma Iron in β-Thalassaemia/Hb E and Haemoglobin H Diseases

Non-transferrin plasma iron concentrations were determined in 45 normal controls and in 37 patients with Hb H disease and 104 patients with β-thalassaemia/Hb E disease. This revealed that non-transferrin plasma iron exists in cases with severe iron overload, more striking in β-thalassaemia/Hb E than in Hb H disease. Non-transferrin plasma iron is associated with higher transferrin iron saturation and higher plasma ferritin levels. The most striking finding was the significantly higher non-transferrin plasma iron in splenectomized patients with β-thalassaemia/Hb E disease than in the non-splenectomized patients. In view of the potential toxicity of non-transferrin iron, this fraction of iron may be responsible for tissue damage in these patients especially after splenectomy.     

Transferrin synthesis by mouse lymph node and peritoneal macrophages: iron content and effect on lymphocyte proliferation

Transferrin is an essential requirement for lymphocyte proliferation, because it supplies activated lymphocytes with iron needed for cell proliferation. However, during inflammation or an immune response, the iron content of circulating transferrin, which is of hepatic origin, decreases. It is hypothesized that activated lymphocytes may therefore obtain transferrin-iron from an alternative source, and we have investigated the possibility that transferrin is synthesized locally in lymphoid tissues. It was found that lymph node cells from mice stimulated in vivo with Freund's complete adjuvant were able to synthesize transferrin, and this was because of the macrophage rather than the lymphocyte population. Transferrin synthesized by mouse lymph node or peritoneal macrophages contained iron and was able to promote mouse lymphocyte proliferation. Peritoneal macrophages activated in vivo synthesized more transferrin, released more transferrin-bound iron, and were more effective than resident macrophages at enhancing lymphocyte proliferation. These results suggest that transferrin synthesized by macrophages acts in a paracrine manner to support lymphocyte proliferation, thus eliminating possible detrimental effect of hypoferremia on the immune system.     

HB Kaohsiung or New York: A T→A Substitution at Codon 113 of the β-Globin Chain Creates an Alu i Cutting Site

Epidemiological studies and experimental data suggest iron involvement in atherosclerosis. The relation between iron and atherosclerosis is complex and remains contradictory. In thalassemia patients, non transferrin bound iron (NTBI) and free hemoglobin (Hb) are present in plasma and may accelerate atherogenesis, but its progression may be inhibited by iron chelators.

The mechanism whereby iron may stimulate atherogenesis has been intensively investigated. Non transferrin bound iron and sera from subjects with hemochromatosis induced endothelial activation with expression of vascular adhesion molecules and endothelial inflammatory chemokines. Such events could be inhibited by iron chelators and oxygen radical scavengers with intracellular activity. Iron chelators may be effective in preventing vascular damage in patients with high concentrations of NTBI as found in thalassemia.     

Iron deficiency anaemia revisited

Iron deficiency anaemia is a global health concern affecting children, women and the elderly, whilst also being a common comorbidity in multiple medical conditions. The aetiology is variable and attributed to several risk factors decreasing iron intake and absorption or increasing demand and loss, with multiple aetiologies often coexisting in an individual patient. Although presenting symptoms may be nonspecific, there is emerging evidence on the detrimental effects of iron deficiency anaemia on clinical outcomes across several medical conditions. Increased awareness about the consequences and prevalence of iron deficiency anaemia can aid early detection and management. Diagnosis can be easily made by measurement of haemoglobin and serum ferritin levels, whilst in chronic inflammatory conditions, diagnosis may be more challenging and necessitates consideration of higher serum ferritin thresholds and evaluation of transferrin saturation. Oral and intravenous formulations of iron supplementation are available, and several patient and disease‐related factors need to be considered before management decisions are made. This review provides recent updates and guidance on the diagnosis and management of iron deficiency anaemia in multiple clinical settings.     

The behavior of transferrin iron in the rat

The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.