J.P. van Dijk

The biology of transferrin

The chemistry and molecular biology of transferrin is discussed. The discussion covers the genetic control of transferrin synthesis, its intracellular synthesis, intra- and extracellular transport, and its interaction with transferrin receptors. The role of transferrin in iron metabolism is evaluated, both with regard to iron uptake by transferrin as to iron uptake from transferrin by different cells. The knowledge on the biochemical mechanisms involved in iron uptake is presented, with special reference to the triple role of the acidification of endocytotic vesicles. Apart from its traditional role in iron metabolism, transferrin acts as a growth factor. A distinction of two groups of growth-stimulating properties of transferrin has been made. As an early effect, membranous and intracellular changes are initiated, possibly based on electrochemical effects on the cell. The late effects seem to relate to its role in iron transport. Interestingly, the early growth stimulating effects can be segregated from the former function of transferrin and strictly speaking neither depend on iron nor on the transferrin molecule itself. Also the trophic effect of transferrin on several cell types has been described. Hypotheses concerning the biochemical basis of this effect are presented and within this context a new hypothesis on the differential occupation of iron binding sites of serum transferrin is forwarded. Examples of the applicability of present understanding of the biology of transferrin in clinical settings are presented.

Review article: Regulatory aspects of placental iron transfer—a comparative study☆

Abstract Receptor-mediated endocytosis is generally assumed to be the process by which the haemochrial placenta takes up iron from transferrin. The involvement of an additional non-endocytic process cannot, however, be excluded. It appears from a study of iron transport mechanisms and of the maturation of the transfer process that placental ferritin is involved in the transfer of iron from mother to fetus. The metabolic relationship between the ferritin pool and the placental transfer pool remains to be elucidated. There is no evidence for short-term regulation of placental transfer capacity in response to changes in the maternal iron supply or to changes in the trophoblastic iron content. This cannot yet be said of fetal feedback control of placental iron uptake because the experiments performed so far do not permit conclusions on this point. The capacity for iron uptake and transfer seems to increase in accordance with the ontogenetically determined placental growth pattern. This does not exclude long-range adaptive modifications of the transfer capacity in response to early maternal or fetal disturbances. The results obtained from studying placental maturation suggest that a possible long-term regulatory interaction between growing placenta and fetus may occur. Clinical evidence so far is inconclusive. The relatively moderate reductions in the fetal iron stores which are generally associated with severe iron-deficient pregnancies might be seen as an argument in favour of long-term placental control. The marked impact of pregnancy on maternal iron metabolism in rodents, as compared to other mammals, is possibly met by means of direct placental control of mucosal iron uptake. In primates, mucosal iron uptake during pregnancy seems to be governed by factors related to systemic iron deficiency only.

Iron Uptake in Blood‐Brain Barrier Endothelial Cells Cultured in Iron‐Depleted and Iron‐Enriched Media

Abstract: Iron is essential in the cellular metabolism of all mammalian tissues, including the brain. Intracerebral iron concentrations vary with age and in several (neurological) diseases. Although it is evident that endothelial cells lining the capillaries in the brain are of importance, factors governing the regulation of intracerebral iron concentration are unknown. To investigate the role of blood‐brain barrier endothelial cells in cerebral iron regulation, primary cultures of porcine blood‐brain barrier endothelial cells were grown in either iron‐enriched or iron‐depleted medium. Iron‐enriched cells showed a reduction in surface‐bound and total transferrin receptor numbers compared with iron‐depleted cells. Transferrin receptor kinetics showed that the transferrin receptor internalization rate in iron‐enriched cultures was higher, whereas the transferrin receptor externalization rate in iron‐enriched cultures was lower than the rate in iron‐depleted cultures. Moreover, blood‐brain barrier endothelial cells cultured in iron‐enriched medium were able to accumulate more iron intracellularly, which underlines our kinetic data on transferrin receptors. Our results agree with histopathological studies on brain tissue of patients with hemochromatosis, suggesting that at high peripheral iron concentrations, the rate of iron transport across the blood‐brain barrier endothelial cells is to some extent proportional to the peripheral iron concentration.

Iron metabolism in the tench (Tinca tinca L.)

SummaryData are presented on iron content and distribution in several fresh and salt water fish species and are compared with those in man and rat. Iron metabolism was studied in the tench by means of intracardiac administration of tench plasma labelled with59Fe(III). The disappearance of plasma iron and the role which the different organs and tissues play in the clearance of plasma iron were studied. A method is presented for calculating internal iron kinetics. Iron transport from the plasma to the erythropoietic system equals the iron transport to the non-erythropoietic organs. To explain this high non-erythropoietic iron transport it is postulated that most of the iron transported to the non-erythropoietic organs will be excreted. The liver-bile system forms an important excretory route. In case of a sudden (erythropoietic) demand for iron the direction of the plasma iron transport may be switched from the liver to the erythropoietic organs.

The appearance of transferrin receptors on cultured human cytotrophoblast and in vitro-formed syncytiotrophoblast

Using trypsinization and Percoll gradient centrifugation, mononuclear cell populations were prepared from human term placentae. These populations consist mainly of cytotrophoblast, which probably lacks transferrin receptors. In vitro, transferrin receptors appear on these mononuclear cells and on syncytiotrophoblast formed in vitro, independently, of cellular fusion and proliferative state.

Transferrin receptor expression and the regulation of placental iron uptake

Placental transferrin receptors, located at the apical side of syncytiotrophoblast, mediate placental iron uptake. Regulation of transferrin receptors on the fetal-maternal exchange area could be a major determinant in the regulation of trans-placental iron transport.Transferrin receptor expression in cultured human term cytotrophoblasts is on a much lower level than in choriocarcinoma cells, with a higher proportion of receptors located on the cell surface. Differentiation of cells, either due to longer culture periods or to 8-bromo-cAMP treatment does not lead to an increase of transferrin receptor expression. In vitro, the level of expression is largely regulated by the cellular density in the culture dishes. Low cellular occupancy of the dish leads to a high level of transferrin receptors. Treatment with iron-sources results in a down regulation of transferrin receptors.Thus, though the level of transferrin receptors in cultured normal trophoblast is at a constant level, unaffected by differentiation, high levels of maternal transferrin-iron availability can lead to a decrease in placental iron uptake. This feed-back mechanism makes placental iron uptake independent of maternal iron stores.

Quantification of different transferrin receptor pools in primary cultures of porcine blood-brain barrier endothelial cells.

Abstract: Distribution of iron in the brain varies with region, cell type, and age. Furthermore, some neurological diseases are accompanied by an abnormal accumulation of iron in specific areas of the CNS. These findings implicate a mobile intracerebral iron pool; however, transport of iron across the blood‐brain barrier and its regulation are largely unknown. In an extensive series of experiments in primary cultures of porcine blood‐brain barrier endothelial cells, we separately quantified surface‐bound and total cellular transferrin receptor pools. Although 90% of all transferrin receptors were located inside the cell, only 10% of these intracellular receptors actively took part in the endocytic cycle. This large “inactive” intracellular transferrin receptor pool could either function as a storage site for spare receptors or be activated by the cell to increase its capacity for iron transport. Data were corrected for nonspecific binding by a separate biochemical assessment using a 100‐fold excess of unlabeled ligand. Data were also analyzed in a nonlinear curve‐fit program. This resulted in a less elaborate and less biased estimate of nonspecific binding.

Regulation of transferrin receptor synthesis by human cytotrophoblast cells in culture.

The aim of this study was to examine the capacity of the syncytiotrophoblast to regulate transferrin receptor (TfR) synthesis in response to modulations in maternal iron supply. The model used was the primary trophoblast cell culture. Trophoblast cells isolated from term human placentas were cultured in iron-poor (Medium 199), iron-depleted (desferrioxamine (DFO)) and iron-supplemented (diferric transferrin (hTf-2Fe), ferric ammonium citrate (FAC) medium. TfR synthesis was reduced in response to hTf-2Fe supplementation. FAC did not modulate TfR synthesis. Iron deprivation by DFO resulted in clear stimulation of TfR synthesis. These results show that the differentiating trophoblast cells respond to pertubations in the (transferrin-mediated) iron supply by adjustments in the rate of TfR synthesis. Taking syncytiotrophoblast in culture as model for the maternal/fetal interface in vivo, our results would suggest that the placenta is able to make short term adjustments of the capacity for iron uptake.

Accumulation and release of iron in polarly and non-polarly cultured trophoblast cells isolated from human term placentas

We investigated the usefulness of membrane grown human term trophoblast cells in transferrin-mediated iron transfer studies. We showed that diferric transferrin is taken up both at the microvillous and at the basal membrane by means of receptor-mediated endocytosis. Uptake from the microvillous side is predominant. This corresponded with a much higher expression of transferrin receptors at the microvillous membrane as compared to the basal one. Iron appeared to accumulate in the cell. Accumulation was higher when transferrin was supplied at the microvillous side. Transfer of iron could not be assessed because uptake of transferrin by the cells was much less than passive diffusion of transferrin through the cell-free filter. The observation of iron accumulation was unexpected for a transfer epithelium. Could it be that part of the iron taken up by the cells is rapidly released whereas the remaining part accumulates? In this case the rate of iron uptake should be higher than the rate of iron accumulation. This question was assessed with non-polarly cultured trophoblast cells. We showed that like in polar cells iron accumulated in ferritin. A new experimental design enabled us to demonstrate that indeed the rate of transferrin-mediated iron is in excess over iron accumulation. We thus provide evidence for a mechanism that enables rapid transfer of iron across the syncytiotrophoblast cell layer.

Binding of human isotransferrin variants to microvillous and basal membrane vesicles from human term placenta

Transferrin (Tf)-dependent iron transfer from mother to fetus is mediated by Tf receptors (TfRs) which are present on both microvillous and basal membranes of human placental syncytiotrophoblast. We used microvillous and basal membrane vesicles, both isolated from the same human term placenta, to investigate the binding of [125I]-labelled diferric bi-bi antennary tetra-sialo Tf (bb Tf), bi-tri-antennary penta-sialo Tf (bt Tf) and tri-tri-antennary hexa-sialo Tf (tt Tf). To diminish the effect of endogenous Tf, membrane vesicles were washed before binding of [125I]-Tf. The number of TfRs on microvillous membranes was 6.1 +/- 2.4 (mean +/- s.d., n = 15) times higher than that on basal membranes, whereas the affinity of TfRs on basal membranes was 3.9 +/- 0.4 (mean +/- s.d., n = 15) times higher than that of TfRs on microvillous membranes, irrespective the isoTf used. The affinity constants of TfRs on both microvillous and basal membranes were higher for bb Tf than for bt Tf and higher for bt Tf than for tt Tf. However, these latter differences were rather small and probably not of physiological importance.