Conservation of copper-transporting P(IB)-type ATPase function

Copper-transporting P(IB)-type ATPases are highly conserved, and while unicellular eukaryotes and invertebrates have only one, a gene duplication has occurred during vertebrate evolution. Copper-induced trafficking of mammalian ATP7A and ATP7B from the trans-Golgi Network towards the plasma membrane is critical for their role in copper homeostasis. In polarized epithelial cells ATP7A and ATP7B traffic towards the basolateral and apical membranes respectively. We examined the localization and function of DmATP7, the single Drosophila melanogaster orthologue, in cultured D. melanogaster and mammalian cells to explore the conservation of P(IB)-type ATPase function. Comparative genomic analysis demonstrated motifs involved in basolateral targeting and retention of ATP7A were conserved in DmATP7, whereas ATP7B targeting motifs were not. DmATP7 expression was able to correct the copper hyper-accumulation phenotype of cultured fibroblasts from a Menkes disease patient expressing a null ATP7A allele. DmATP7 was able to transport copper to the cupro-enzyme tyrosinase and under elevated copper conditions DmATP7 was able to traffic towards the plasma membrane and efflux copper, essentially phenocopying ATP7A. When expressed in polarized Madin-Darby Canine Kidney cells, DmATP7 translocated towards the basolateral membrane when exposed to elevated copper, similar to ATP7A. These results demonstrate DmATP7 is able to functionally compensate for the absence of ATP7A, with important trafficking motifs conserved in these distantly related orthologues.     

Human copper transporter hCTR1 mediates basolateral uptake of copper into enterocytes: implications for copper homeostasis

Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal Madin-Darby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.     

Menkes Copper ATPase (Atp7a) is a novel metal-responsive gene in rat duodenum, and immunoreactive protein is present on brush-border and basolateral membrane domains

We previously noted strong induction of genes related to intestinal copper homeostasis (Menkes Copper ATPase (Atp7a) and metallothionein) in the duodenal epithelium of iron-deficient rats across several stages of postnatal development (Collins, J. F., Franck, C. A., Kowdley, K. V., and Ghishan, F. K. (2005) Am. J. Physiol., 288, G964-G971). We now report significant copper loading in the livers and intestines of iron-deficient rats. These findings are consistent with the hypothesis that there is increased intestinal copper transport during iron deficiency. We additionally found that hepatic Atp7b gene expression does not change with iron deficiency, suggesting that liver copper excretion is not altered. We have developed polyclonal antibodies against rat ATP7A, and we demonstrate the specificity of the immunogenic reaction. We show that the ATP7A protein is present on apical domains of duodenal enterocytes in control rats and on brush-border and basolateral membrane domains in iron-deprived rats. This localization is surprising, as previous in vitro studies have suggested that ATP7A traffics between the trans-Golgi network and the basolateral membrane. We further demonstrate that ATP7A protein levels are dramatically increased in brush-border and basolateral membrane vesicles isolated from iron-deficient rats. Other experiments show that iron refeeding partially corrects the hematological abnormalities seen in iron-deficient rats but that it does not ameliorate ATP7A protein induction, suggesting that Atp7a does not respond to intracellular iron levels. We conclude that ATP7A is involved in copper loading observed during iron deficiency and that increased intestinal copper transport is of physiological relevance, as copper plays important roles in overall body iron homeostasis.     

A polarized human endometrial cell line that binds and transports polymeric IgA

Summary We have demonstrated that a human endometrial cell line, HEC-1, maintains a high transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surfaces of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.     

Cell-Specific Trafficking Suggests a new role for Renal ATP7B in the Intracellular Copper Storage

Human Cu-ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney-specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not on account of the loss of a kinase-mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2–3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell-specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage.     

N-glycans are not a universal signal for apical sorting of secretory proteins.

In MDCK cells, N-glycans have been shown to determine the sorting of secretory proteins and membrane proteins to the apical domain in the absence of a dominant basolateral targeting signal. We have examined the sorting of endogenous proteins in ECV304 cells in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation. A prominent apically secreted protein of 71 kDa was not N-glycosylated and continued to be secreted apically in the presence of tunicamycin. In contrast, other endogenous proteins that were N-glycosylated were secreted preferentially into the basolateral medium or without polarity. When rat growth hormone was expressed in MDCK and ECV304 cells, we observed 65 and 94% of the secretion to the basolateral medium, respectively. Introduction of a single N-glycan caused 83% of the growth hormone to be secreted at the apical surface in MDCK cells but had no significant effect on the polarity of secretion of growth hormone in ECV304 cells. These results indicate that not all cell lines recognise N-glycans as a signal for apical sorting and raises the possibility of using ECV304 cells as a model system for analysis of apical sorting molecules.     

Expression of ATP7B in normal human liver

ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.     

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.     

ATP7B expression in human breast epithelial cells is mediated by lactational hormones.

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper ((64)Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.     

Identification of differentially expressed genes in response to dietary iron deprivation in rat duodenum

We sought to identify novel genes involved in intestinal iron absorption by inducing iron deficiency in rats during postnatal development from the suckling period through adulthood. We then performed comparative gene chip analyses (RAE230A and RAE230B chips; Affymetrix) with cRNA derived from duodenal mucosa. Real-time PCR was used to confirm changes in gene expression. Genes encoding the apical iron transport-related proteins [divalent metal transporter 1 (DMT1) and duodenal cytochrome b] were strongly induced at all ages studied, whereas increases in mRNA encoding the basolateral proteins iron-regulated gene 1 and hephaestin were observed only by real-time PCR. In addition, transferrin receptor 1 and heme oxygenase 1 were induced. We also identified induction of novel genes not previously associated with intestinal iron transport. The Menkes copper ATPase (ATP7a) and metallothionein were strongly induced at all ages studied, suggesting increased copper absorption by enterocytes during iron deficiency. We also found significantly increased liver copper levels in 7- to 12-wk-old iron-deficient rats. Also upregulated at most ages examined were the sodium-dependent vitamin C transporter, tripartite motif protein 27, aquaporin 4, lipocalin-interacting membrane receptor, and the breast cancer-resistance protein (ABCG2). Some genes also showed decreased expression with iron deprivation, including several membrane transporters, metabolic enzymes, and genes involved in the oxidative stress response. We speculate that dietary iron deprivation leads to increased intestinal copper absorption via DMT1 on the brush-border membrane and the Menkes copper ATPase on the basolateral membrane. These findings may thus explain copper loading in the iron-deficient state. We also demonstrate that many other novel genes may be differentially regulated in the setting of iron deprivation.     

Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.     

Polarized transport of the polymeric immunoglobulin receptor in transfected rabbit mammary epithelial cells

A cDNA for the rabbit low Mr polymeric immunoglobulin (poly-Ig) receptor was expressed in an immortalized rabbit mammary cell line. The intracellular routing of the receptor and its cell surface expression was analyzed in stably transfected cells grown on permeable supports. Initially the cells formed a monolayer with no transmural electrical resistance. All monolayer cells expressed the poly-Ig receptor and cytokeratin 7 filaments characteristic of luminal mammary cells but absent in myoepithelial cells. Within 7 d in culture, the cells underwent cytodifferentiation and formed a bilayer with a transepithelial electrical resistance of approximately 500 omega x cm2. Upper layer cells formed tight junctions with adjacent cells and gap junctions with basal cells. Expression of the poly-Ig receptor and cytokeratin 7 was restricted to the cells from the upper layer. The kinetics of receptor biosynthesis and processing was similar to that reported for rabbit mammary gland and rat liver. The receptor was cleaved at the apical cell surface and release of secretory component into the apical medium occurred with a half-time of approximately 2 h. Selective cell surface trypsinization combined with pulse-chase experiments served to determine at which cell surface domain newly synthesized receptor appeared first. The receptor was digested with a half-time of approximately 60 min with trypsin present in the basolateral medium and 90 min with apical trypsin. These data are consistent with selective targeting of newly synthesized receptor to the basolateral surface. The results indicate that transcytosis of the receptor from basolateral to apical membrane in the presence or the absence of its ligand requires approximately 30 min. Cleavage of the receptor by endogenous protease is not concomitant with its appearance at the apical surface, but requires additional time, thus explaining the presence of intact receptor on the apical membrane.     

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.     

Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B

The copper-transporting P(1B)-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases.     

Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes

Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu‐directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF‐B and in the liver in vivo. Copper (10 µm ) caused ATP7B to exit the trans‐Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton‐pump inhibitor bafilomycin‐A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu‐stimulated) case. Overall, loss of acidification‐impaired Cu‐regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane.      

Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B,as Revealed by Nanobody Binding

The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell.     

Signals regulating trafficking of Menkes (MNK; ATP7A) copper-translocating P-type ATPase in polarized MDCK cells

The Menkes protein (MNK; ATP7A) functions as a transmembrane copper-translocating P-type ATPase and plays a vital role in systemic copper absorption in the gut and copper reabsorption in the kidney. Polarized epithelial cells such as Madin-Darby canine kidney (MDCK) cells are a physiologically relevant model for systemic copper absorption and reabsorption in vivo. In this study, cultured MDCK cells were used to characterize MNK trafficking and enabled the identification of signaling motifs required to target the protein to specific membranes. Using confocal laser scanning microscopy and surface biotinylation we demonstrate that MNK relocalizes from the Golgi to the basolateral (BL) membrane under elevated copper conditions. As previously shown in nonpolarized cells, the metal binding sites in the NH₂-terminal domain of MNK were found to be required for copper-regulated trafficking from the Golgi to the plasma membrane. These data provide molecular evidence that is consistent with the presumed role of this protein in systemic copper absorption in the gut and reabsorption in the kidney. Using site-directed mutagenesis, we identified a dileucine motif proximal to the COOH terminus of MNK that was critical for correctly targeting the protein to the BL membrane and a putative PDZ target motif that was required for localization at the BL membrane in elevated copper. Menkes disease; PDZ; copper; trafficking     

Distinct functional roles for the Menkes and Wilson copper translocating P-type ATPases in human placental cells.

BACKGROUND/AIMS: The copper transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND) are essential for normal copper transport in the human body. The placenta is the key organ in copper supply to the fetus during pregnancy and it is one of the few organs in the body to express both of the ATPases. The placenta therefore provides a unique opportunity to elucidate the specific roles of these transporters within the one cell type. METHODS/RESULTS: Using polarized placental Jeg-3 cells, siRNA technology and radio-labelled 64Cu transport assays, MNK and WND were shown to have distinct roles in the vectorial transport of copper. MNK transported copper from the cell via the basolateral membrane and in contrast, WND transported copper from the apical membrane. Inactivation of MNK resulted in decreased activity of two important cuproenzymes, lysyl oxidase and Cu/Zn-superoxide dismutase. CONCLUSIONS: Overall, these results provide definitive evidence for distinct roles of MNK and WND in the human placenta, and are consistent with a role for MNK in the transport of copper into the fetal circulation, and through delivery of copper to placental cuproenzymes, whilst WND contributes to the maintenance of placental copper homeostasis by transporting copper to the maternal circulation.     

Effects of copper and ceruloplasmin on iron transport in the Caco 2 cell intestinal model

Previous studies have implicated copper proteins, including ceruloplasmin, in intestinal iron transport. Polarized Caco2 cells with tight junctions were used to examine the possibilities that (a) ceruloplasmin promotes iron absorption by enhancing release at the basolateral cell surface and (b) copper deficiency reduces intestinal iron transport. Iron uptake and overall transport were followed for 90 min with 1 &mgr;M 59Fe(II) applied to the apical surface of Caco2 cell monolayers. Apotransferrin (38 &mgr;M) was in the basolateral chamber. Induction of iron deficiency with desferrioxamine (100 &mgr;M; 18 h) markedly increased uptake and overall transport of iron. Uptake increased from about 20% to about 65% of dose, and overall 59Fe transport from <1% to 60% of dose. On the basis of actual iron released into the basal chamber (measured with bathophenanthroline), transport increased 8-fold. Desferrioxamine pretreatment reduced cellular Fe by 55%. The addition of freshly isolated, enzymatically active human ceruloplasmin to the basolateral chamber during absorption had no effect on uptake or transport of iron by the cells. Unexpectedly, pretreatment with three different chelators of copper (18 h), which reduced cellular levels about 40%, more than doubled iron uptake and raised overall transport to 20%. This was so, whether or not cells were also made iron deficient with desferrioxamine. Acute addition of 1 &mgr;M Cu(II) to the apical chamber had no significant effect upon iron uptake, retention, or transport in iron deficient or normal cells, in the presence of absence of ascorbate. We conclude that intestinal absorption of Fe(II) is unlikely to depend upon plasma ceruloplasmin, and that cuproproteins involved in this form of iron transport must be binding copper tightly.     

Trafficking of the copper-ATPases, ATP7A and ATP7B: role in copper homeostasis

Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.