Ferroportin 1 and hephaestin expression in BeWo cell line with different iron treatment

The process of placental iron transfer is an important physiological process during pregnancy. However, the molecular mechanism of placental iron transport has not been completely elucidated until now. Ferroportin 1 (FPN1) and hephaestin (Heph) have been identified as the important molecules involved in duodenal iron export. However, whether they participate in the placental iron efflux has been undefined until now. In this study, the BeWo cells were treated with desferrioxamine and Holo-transferrin human in different concentrations and harvested at 48 and 72 h. The mRNA expression of FPN1 and Heph was detected with quantitative real-time polymerase chain reaction, and the protein expression was detected with western blots. The results showed an up-regulated FPN1 expression with desferrioxamine treatment and down-regulated expression with Holo-transferrin human supplementation. However, the change of FPN1 expression at protein level was limited. Heph expression enhanced when cells were treated with desferrioxamine although the quantity of Heph expression was low. Heph expression showed no significant change with Holo-transferrin human supplementation. It indicates that FPN1 may participate in placental iron transport, and placental FPN1 expression is obviously not dependent on the iron regular element/iron regular protein regulation. An alternatively spliced FPN1 isoform that lacks an iron regular element may be the predominant expression in BeWo cells. It also demonstrates that Heph is active in placenta but may not play a key role in placental iron transport because it is not the main part of placental copper oxidase.     

Iron feeding induces ferroportin 1 and hephaestin migration and interaction in rat duodenal epithelium

Intestinal iron absorption involves proteins located in the brush border membrane (BBM), cytoplasm, and basolateral membrane (BLM) of duodenal enterocytes. Ferroportin 1 (FPN1) and hephaestin (Heph) are necessary for transport of iron out of enterocytes, but it is not known whether these two proteins interact during iron absorption. We first examined colocalization of the proteins by cotransfection of HEK293 cells with pDsRed-FPN1 with pEmGFP-Heph or with the COOH-terminal truncated pEmGFP-HephDelta43 or -HephDelta685 and found that FPN1 and Heph with or without the COOH terminus colocalized. In rat duodenal enterocytes, within 1 h of iron feeding prominent migration of FPN1 from the apical subterminal zone to the basal subnuclear zone of the BLM occurred and increased to at least 4 h after feeding. Heph exhibited a similar though less prominent migration after iron ingestion. Analysis using rat duodenal epithelial cell sheets demonstrated that 1) by velocity sedimentation ultracentrifugation, FPN1 and Heph occupied vesicles of different sizes prior to iron feeding and migrated to similar fractions 1 h after iron feeding; 2) by blue native/SDS-PAGE, FPN1, and Heph interacted to form two complexes, one containing dimeric FPN1 and intact Heph and the other consisting of monomeric FPN1 and a Heph fragment; and 3) by immunoprecipitation, anti-Heph or anti-FPN1 antiserum coimmunoprecipitated FPN1 and Heph. Thus the data indicate that FPN1 and Heph migrate and interact during iron feeding and suggest that dimeric FPN1 is associated with intact Heph.     

Immunoreactive hephaestin and ferroxidase activity are present in the cytosolic fraction of rat enterocytes

Discovered over a decade ago, hephaestin (Heph) has been implicated as a ferroxidase (FOX) vital for intestinal iron absorption. Stringent structural or kinetic data derived from purified, native protein is however lacking, leading to the hypothesis that an alternate, undiscovered form of Heph could exist in mammalian enterocytes. This possibility was tested using laboratory rodent and cell culture models. Cytosolic and membrane fractions were obtained from rat enterocytes and purity of the fractions was assessed. Western blot analyses revealed Heph in cytosol obtained by three different methods, ruling out the possibility of a method-induced artifact being the major contributor to this observation. Absence of two different membrane-proteins, ferroportin 1 and Menke's copper ATPase in cytosol, and the absence of lipids in representative cytosolic samples tested by thin layer chromatography, eliminated significant membrane contamination of cytosol. Further, immunohisto- and immunocyto-chemical analyses identified Heph in rat enterocytes and in two intestinal epithelial cell lines, IEC-6 and Caco-2, intracellularly. Additionally, cytosolic Heph increased upon iron-deprivation but more important, decreased significantly upon copper-deprivation, mimicking the response of membrane-bound Heph. Moreover, FOX activity was present in rat cytosol, and was partly inhibited by anti-Heph antibody. Finally, lack of immunodetectable ceruloplasmin (Cp) by western blot precluded Cp as an underlying cause of this activity. These data demonstrate that rat enterocytes contain a soluble/cytosolic form of Heph possibly contributing to the observed FOX activity.     

Age-dependent expression of hephaestin in the brain of ceruloplasmin-deficient mice

Aceruloplasminemia is an autosomal recessive disorder caused by mutations in the ceruloplasmin (CP) gene. It is characterized by iron accumulation in the brain and in visceral organs. However, little is known about the mechanism of iron transport in these regions. Adult CP null (CP^−/−) mice show increased iron deposition in several regions of brain, such as the cerebellum and brainstem. In this study, we investigated the expression of the ceruloplasmin homolog hephaestin (Heph) in the brain of CP^−/− mice as a function of age. In the cerebral cortex and caudate putamen of 80-week-old CP^−/− mice, the expression of Heph increased significantly whilst iron levels remain normal [Patel BN, Dunn RJ, Jeong SY, Zhu Q, Julien JP, David S. Ceruloplasmin regulates iron levels in the CNS and prevents free radical injury. J Neurosci 2002;22(15):6578–6], indicating that Heph might compensate for the loss of CP. In contrast, the substantia nigra and cerebellum of 80-week-old CP^−/− mice accumulate iron but do not express high levels or significant decrease of Heph, suggesting that Heph does not replace CP in these regions. These data suggest that Heph may compensate for the loss of CP in a region-specific manner.     

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.     

Colocalization of ferroportin-1 with hephaestin on the basolateral membrane of human intestinal absorptive cells

An iron exporter ferroportin-1 (FPN-1) and a multi-copper oxidase hephaestin (Heph) are predicted to be expressed on the basolateral membrane of the enterocyte and involved in the processes of iron export across the basolateral membrane of the enterocyte. However, it is not clear where these proteins are exactly located in the intestinal absorptive cell. We examined cellular localization of FPN-1 and Heph in the intestinal absorptive cells using the fully differentiated Caco-2 cells. Confocal microscope study showed that FPN-1 and Heph are located on the basolateral membrane and they are associated with the transferrin receptor (TfR) in fully differentiated Caco-2 cells grown on microporous membrane inserts. However, Heph protein was not detected in the crypt cell-like proliferating Caco-2 cell. In stably transfected human intestinal absorptive cells expressing human FPN-1 modified by the addition of GFP at the C-terminus, we show that FPN-1-GFP is located on the basolateral membrane and it is associated with Heph suggesting the possibility that FPN-1 might associate and interact with Heph in the process of iron exit across the basolateral membrane of intestinal absorptive cell.     

Haemopexin affects iron distribution and ferritin expression in mouse brain

Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme‐mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer’s disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron‐loaded oligodendrocytes in the basal ganglia and thalamus of Hx‐null mice compared to wild‐type controls. Interestingly, there was no increase in H‐ and L‐ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer’s disease and Parkinson’s disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin‐positive cells was observed in the cerebral cortex of Hx‐null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu–Zn superoxide dismutase‐1 expression were higher in the brain of Hx‐null mice than in that of wild‐type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron‐related neurodegenerative diseases.     

Brain endothelial barrier passage by monocytes is controlled by the endothelin system

Homeostasis of the brain is dependent on the blood-brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ET(B)) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases.     

Brain iron transport

Brain iron is a crucial participant and regulator of normal physiological activity. However, excess iron is involved in the formation of free radicals, and has been associated with oxidative damage to neuronal and other brain cells. Abnormally high brain iron levels have been observed in various neurodegenerative diseases, including neurodegeneration with brain iron accumulation, Alzheimer's disease, Parkinson's disease and Huntington's disease. However, the key question of why iron levels increase in the relevant regions of the brain remains to be answered. A full understanding of the homeostatic mechanisms involved in brain iron transport and metabolism is therefore critical not only for elucidating the pathophysiological mechanisms responsible for excess iron accumulation in the brain but also for developing pharmacological interventions to disrupt the chain of pathological events occurring in these neurodegenerative diseases. Numerous studies have been conducted, but to date no effort to synthesize these studies and ideas into a systematic and coherent summary has been made, especially concerning iron transport across the luminal (apical) membrane of the capillary endothelium and the membranes of different brain cell types. Herein, we review key findings on brain iron transport, highlighting the mechanisms involved in iron transport across the luminal (apical) as well as the abluminal (basal) membrane of the blood–brain barrier, the blood–cerebrospinal fluid barrier, and iron uptake and release in neurons, oligodendrocytes, astrocytes and microglia within the brain. We offer suggestions for addressing the many important gaps in our understanding of this important topic, and provide new insights into the potential causes of abnormally increased iron levels in regions of the brain in neurodegenerative disorders.     

The iron regulatory hormone hepcidin reduces ferroportin 1 content and iron release in H9C2 cardiomyocytes

Iron plays a key pathophysiological role in a number of cardiac diseases. Studies on the mechanisms of heart iron homeostasis are therefore crucial for understanding the causes of excessive heart iron. In addition to iron uptake, cellular iron balance in the heart also depends on iron export. We provided evidence for the existence of iron exporter ferroportin 1 (Fpn1) in the heart in a recent study. The presence of hepcidin, a recently discovered iron regulatory hormone, was also confirmed in the heart recently. Based on these findings and the inhibiting role of hepcidin on Fpn1 in other tissues, we speculated that hepcidin might be able to bind with, internalize and degrade Fpn1 and then decrease iron export in heart cells, leading to an abnormal increase in heart iron and iron mediated cell injury. We therefore investigated the effects of hepcidin on the contents of Fpn1 and iron release in H9C2 cardiomyocyte cell line. We demonstrated that hepcidin has the ability to reduce Fpn1 content as well as iron release in this cell. The similar regulation patterns of hepcidin on the Fpn1 and iron release suggested that the decreased iron release resulted from the decreased content of Fpn1 induced by hepcidin. We also found that hepcidin has no significant effects on ceruloplasmin (CP) and hephaestin (Heph) — two proteins required for iron release from mammalian cells. The data imply that Fpn1, rather than Heph and CP, is the limited factor in the regulation of iron release from heart cells under physiological conditions.     

Effects of electromagnetic pulse exposure on gelatinase of blood–brain barrier in vitro

The biological effects of electromagnetic pulse (EMP) on the brain have been focused on for years. It was reported that gelatinase played an important role in maintaining brain function through regulating permeability in the blood–brain barrier (BBB). To investigate the effects of EMP on gelatinase of BBB, an in vitro BBB model was established using primary cultured rat brain microvascular endothelial cells (BMVEC), astrocytes and half-contact culture of these cells in a transwell chamber. Cultured supernatant and cells were collected at different time points after exposure to EMP (peak intensity 400 kV/m, rise time 10 ns, pulse width 350 ns, 0.5 pps and 200 pulses). Protein levels of cellular gelatinase MMP-2 and MMP-9, and endogenous inhibitor TIMP-1 and TIMP-2 were detected by Western blot. The activity of gelatinase in culture supernatant was detected by gelatin zymography. It was found that compared with the sham-exposed group, the protein level of MMP-2 was significantly increased at 6 h (p < 0.05), and the protein level of its endogenous inhibitor TIMP-2 did not change after EMP exposure. In addition, the protein levels of MMP-9 and its endogenous inhibitor TIMP-1 did not change after EMP exposure. Gelatin zymography results showed that the activity of MMP-2 in the inner pool and the outer pool of the transwell chamber was significantly increased at 6 h after EMP exposure compared with that of the sham group. These results suggested that EMP exposure could affect the expression and activity of MMP-2 in the BBB model.     

Iron Imports. VI. HFE and regulation of intestinal iron absorption

The majority of clinical cases of iron overload is caused by mutations in the HFE gene. However, the role that HFE plays in the physiology of intestinal iron absorption remains enigmatic. Two major models have been proposed: 1) HFE exerts its effects on iron homeostasis indirectly, by modulating the expression of hepcidin; and 2) HFE exerts its effects directly, by changing the iron status (and therefore the iron absorptive activity) of intestinal enterocytes. The first model places the primary role of HFE in the liver (hepatocytes and/or Kupffer cells). The second model places the primary role in the duodenum (crypt cells or villus enterocytes). These models are not mutually exclusive, and it is possible that HFE influences the iron status in each of these cell populations, leading to cell type-specific downstream effects on intestinal iron absorption and body iron distribution.     

The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor-mediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K+ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-beta-cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, alpha(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes alpha(2,3)-, but not with Sambucus nigra agglutinin that recognizes alpha(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal alpha(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.     

In Search of the Astrocytic Factor(s) Modulating Blood–Brain Barrier Functions in Brain Capillary Endothelial Cells In Vitro

Summary 1. The blood–brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). There are various cell types, in particular astrocytes, but also pericytes and neurons, located in close vicinity to the capillary ECs which may influence formation and function of the BBB. Based on this consideration, this paper discusses various aspects of the influence of the surrounding cells on brain capillary ECs with special focus on the role of astrocytes.2. Based on the morphology of the BBB, important aspects of brain EC functions are summarized, such as transport functions and maintenance of low paracellular permeability. Moreover, various facets are discussed with respect to the influence of astrocytes, pericytes, microglia, and neurons on the BBB. Data on the role of glial cells in the ontogenesis of the BBB are presented subsequently. The knowledge on this subject is far from being complete, however, these data imply that the neural/neuronal environment rather than glial cells may be of importance in the maturation of the barrier.3. The role of glial cells in the induction and maintenance of the BBB is discussed under physiological as well as pathological conditions. Although the literature presents manifold evidence for a great variety of effects induced by astroglia, there are also many controversies, which may result from different cellular models and experimental conditions used in the respective studies. Numerous factors secreted by astrocytes have been shown to induce a BBB phenotype. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) is documented in the presence of astrocyte-derived factors, and many studies demonstrate the improvement of physiological parameters, such as increased transendothelial resistance and decreased paracellular permeability, in different in vitro models of the BBB. Moreover, one has to take into account that the interaction of brain ECs and astrocytes is bi-directional, and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction, respectively.4. In conclusion, it is expected that the present and future research focused on molecular mechanisms and signaling pathways will produce new and exciting insights into the complex network of BBB regulation: the cornerstone is laid.This revised article was published online in May 2005 with a February 2005 cover date.     

Inflammation alters the expression of DMT1, FPN1 and hepcidin,and it causes iron accumulation in central nervous system cells

Inflammation and iron accumulation are present in a variety of neurodegenerative diseases that include Alzheimer's disease and Parkinson's disease. The study of the putative association between inflammation and iron accumulation in central nervous system cells is relevant to understand the contribution of these processes to the progression of neuronal death. In this study, we analyzed the effects of the inflammatory cytokines tumor necrosis factor alpha (TNF‐α) and interleukin 6 (IL‐6) and of lipopolysaccharide on total cell iron content and on the expression and abundance of the iron transporters divalent metal transporter 1 (DMT1) and Ferroportin 1 (FPN1) in neurons, astrocytes and microglia obtained from rat brain. Considering previous reports indicating that inflammatory stimuli induce the systemic synthesis of the master iron regulator hepcidin, we identified brain cells that produce hepcidin in response to inflammatory stimuli, as well as hepcidin‐target cells. We found that inflammatory stimuli increased the expression of DMT1 in neurons, astrocytes, and microglia. Inflammatory stimuli also induced the expression of hepcidin in astrocytes and microglia, but not in neurons. Incubation with hepcidin decreased the expression of FPN1 in the three cell types. The net result of these changes was increased iron accumulation in neurons and microglia but not in astrocytes. The data presented here establish for the first time a causal association between inflammation and iron accumulation in brain cells, probably promoted by changes in DMT1 and FPN1 expression and mediated in part by hepcidin. This connection may potentially contribute to the progression of neurodegenerative diseases by enhancing iron‐induced oxidative damage.     

Inhibition of melanoma brain metastasis by targeting melanotransferrin at the cell surface

Brain metastases are a common feature of malignant melanoma and are associated with poor prognosis. Melanotransferrin (MTf), one of several antigens associated with the surface of melanoma cells, has been demonstrated to promote cell invasion. In this study, we investigated the role of membrane‐bound MTf in several of the steps leading to the development of melanoma brain metastasis. Our results indicated that MTf‐positive cells were detected in the brains of nude mice injected intravenously with human melanoma SK‐Mel 28 cells. Moreover, administration of a single dose of a monoclonal antibody (L235) directed against human MTf significantly reduced the development of human melanoma brain metastases in nude mice. The ability of melanoma cells to cross the blood–brain barrier (BBB) in vitro is correlated with their MTf expression levels at the cell surface. Overall, our results indicated that membrane‐bound MTf is a key element in melanoma cell transmigration across the BBB and subsequent brain metastasis. Thus, these data suggest MTf as an attractive target and demonstrate the therapeutic potential of an anti‐MTf mAb for preventing metastatic melanoma.     

sAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin‐targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.     

Transient Expression of Iron Transport Proteins in the Capillary of the Developing Rat Brain

Iron is essential for normal brain function and its uptake in the developing rat brain peaks during the first two weeks after birth, prior to the formation of the blood–brain barrier (BBB). The first step of iron transport from the blood to the brain is transferrin receptor (TfR)-mediated endocytosis in the capillary endothelial cells. However, the subsequent step from the endothelium into interstitium has not been fully described. The goal of this study was to examine the expression of iron transport proteins by immunodetection and RT–PCR in the developing rat brain. Tf and TfR are transiently expressed in perivascular NG2+ cells of the capillary wall during the early postnatal weeks in the rat brain. However, MTP-1 and hephaestin were expressed in endothelial cells, but not in the NG2+ perivascular cells. Immunoblot analysis for these iron transfer proteins in the developing brain generally confirmed the immunochemical findings. Furthermore, the expression of Tf and TfR in the blood vessels precedes its expression in oligodendrocytes, the main iron-storing cells in the vertebrate brain. RT–PCR analysis for the primary culture of endothelial cells and pericytes revealed that Tf and TfR were highly expressed in the pericytes while MTP-1 and hephaestin were expressed in the endothelial cells. The specific expression of Tf and TfR in brain perivascular cells and MTP-1 and hephaestin in endothelial cells suggest the possibility that trafficking of elemental iron through perivascular cells may be instrumental in the distribution of iron in the developing central nervous system.