Copper Regulates Maturation and Expression of an MITF:Tryptase Axis in Mast Cells.

Copper has previously been implicated in the regulation of immune responses, but the impact of this metal on mast cells is poorly understood. In this article, we address this issue and show that copper starvation of mast cells causes increased granule maturation, as indicated by higher proteoglycan content, stronger metachromatic staining, and altered ultrastructure in comparison with nontreated cells, whereas copper overload has the opposite effects. In contrast, copper status did not impact storage of histamine in mast cells, nor did alterations in copper levels affect the ability of mast cells to degranulate in response to IgER cross-linking. A striking finding was decreased tryptase content in mast cells with copper overload, whereas copper starvation increased tryptase content. These effects were associated with corresponding shifts in tryptase mRNA levels, suggesting that copper affects tryptase gene regulation. Mechanistically, we found that alterations in copper status affected the expression of microphthalmia-associated transcription factor, a transcription factor critical for driving tryptase expression. We also found evidence supporting the concept that the effects on microphthalmia-associated transcription factor are dependent on copper-mediated modulation of MAPK signaling. Finally, we show that, in MEDNIK syndrome, a condition associated with low copper levels and a hyperallergenic skin phenotype, including pruritis and dermatitis, the number of tryptase-positive mast cells is increased. Taken together, our findings reveal a hitherto unrecognized role for copper in the regulation of mast cell gene expression and maturation.

Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors.

Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin ß7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function.

Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain.

Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu(+) transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine- and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu(+) transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu(+) and cisplatin acquisition.

Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans.

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain.

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2(-/-) mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.

Intestinal DMT1 is critical for iron absorption in the mouse but is not required for the absorption of copper or manganese.

Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)). DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and (54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc.

Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?

The human copper (Cu) chaperone Atox1 delivers Cu to P1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC) motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.

Charting the travels of copper in eukaryotes from yeast to mammals.

Throughout evolution, all organisms have harnessed the redox properties of copper (Cu) and iron (Fe) as a cofactor or structural determinant of proteins that perform critical functions in biology. At its most sobering stance to Earth's biome, Cu biochemistry allows photosynthetic organisms to harness solar energy and convert it into the organic energy that sustains the existence of all nonphotosynthetic life forms. The conversion of organic energy, in the form of nutrients that include carbohydrates, amino acids and fatty acids, is subsequently released during cellular respiration, itself a Cu-dependent process, and stored as ATP that is used to drive a myriad of critical biological processes such as enzyme-catalyzed biosynthetic processes, transport of cargo around cells and across membranes, and protein degradation. The life-supporting properties of Cu incur a significant challenge to cells that must not only exquisitely balance intracellular Cu concentrations, but also chaperone this redox-active metal from its point of cellular entry to its ultimate destination so as to avert the potential for inappropriate biochemical interactions or generation of damaging reactive oxidative species (ROS). In this review we chart the travels of Cu from the extracellular milieu of fungal and mammalian cells, its path within the cytosol as inferred by the proteins and ligands that escort and deliver Cu to intracellular organelles and protein targets, and its journey throughout the body of mammals. This article is part of a Special Issue entitled: Cell Biology of Metals.

Folates in bread: retention during bread-making and in vitro bioaccessibility.

BACKGROUND: Bread is an important folate source in several countries. However, bread-making was reported to cause losses of endogenous bread folates (approximately 40%) as well as added synthetic folic acid (approximately 30%). Furthermore, the bread matrix is suggested to inhibit absorption of folates. PURPOSE: To (1) estimate retention of both, endogenous folates and synthetic fortificants, during bread-making, (2) assess in vitro folate bioaccessibility from breads and a breakfast meal and (3) assess in vitro folate uptake. METHODS: Retention of folate forms was assessed by preparing fortified (folic acid and [6S]-5-CH(3)-H(4)folate) wholemeal breads and collect samples from dough, proofed dough and the bread. In vitro folate bioaccessibility was assessed using the TNO gastrointestinal model TIM. In vitro folate uptake was assessed using a novel Caco-2 cell/stable isotope model. Folate content in samples was measured using LCMS. RESULTS: Bread-making resulted in losses of 41% for endogenous folates and up to 25 and 65% for folic acid and [6S]-5-CH(3)-H(4)folate fortificant, respectively. 75% of endogenous bread folates and 94% of breakfast folates were bioaccessible as assessed by TIM. From [6S]-5-CH(3)-H(4)folate-fortified bread, relative folate uptake into Caco-2 cells was 71 +/- 11% (P < 0.05) when compared with a standard solution. CONCLUSION: Retention of folic acid fortificant during bread-making was substantially higher compared to retention of [6S]-5-CH(3)-H(4)folate fortificant. Data from the TIM and Caco-2 cell trials suggest an inhibiting effect of the tested bread matrices on in vitro bioaccessibility of folates, whereas folate bioaccessibility from a breakfast meal is almost complete.

Mitogen-Activated Protein Kinase Signaling Regulates Proteoglycan Composition of Mast Cell Secretory Granules.

Mast cells (MCs) are characterized by an abundance of lysosome-like secretory granules filled with immunomodulatory compounds including histamine, cytokines, lysosomal hydrolases, MC-restricted proteases, and serglycin proteoglycans. The latter are essential for promoting the storage of other granule compounds and are built up of the serglycin core protein to which highly sulfated and thereby negatively charged glycosaminoglycan (GAG) side chains of heparin or chondroitin sulfate type are attached. In the search for mechanisms operating in regulating MC granule homeostasis, we here investigated the role of mitogen-activated protein kinase (MAPK) signaling. We show that inhibition of MEK1/2 (a MAPK kinase) leads to increased metachromatic staining of MC granules, indicative of increased proteoglycan content. Indeed, MEK1/2 inhibition caused a profound increase in the expression of the gene coding for the serglycin core protein and of genes coding for various enzymes involved in the biosynthesis/sulfation of the GAGs attached to the serglycin core protein. This was accompanied by corresponding increases in the levels of the respective GAGs. Deletion of the serglycin core protein abrogated the induction of enzymes operative in proteoglycan synthesis, indicating that availability of the serglycin proteoglycan core protein has a regulatory function impacting on the expression of the various serglycin-modifying enzymes. MEK1/2 inhibition also caused a substantial increase in the expression of granule-localized, proteoglycan-binding proteases. Altogether, this study identifies a novel role for MAPK signaling in regulating the content of secretory granules in MCs.

Impact of iron status on cadmium uptake in suckling piglets.

Low iron status is known to increase the uptake of dietary cadmium in both adolescents and adults and there are indications that cadmium is absorbed from the intestine by the two major iron transporters divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1). In addition, it has been suggested that duodenal metallothionein (MT) may limit the transport of cadmium across the intestinal epithelium. The present investigation was undertaken to examine whether iron status influences cadmium absorption in newborns by applying a model of suckling piglets and the possible roles of duodenal DMT1, FPN1 and MT. An oral cadmium dose (20 microg/kg body weight) was given daily for 6 consecutive days on postnatal days (PNDs) 10-15 to iron-deficient or iron-supplemented piglets. The cadmium dose was chosen to keep the cadmium level at a realistically low but still detectable level, and without inducing any adverse health effects in the piglets. As indicators of cadmium uptake, cadmium levels in blood and kidneys were measured on PND 16 by inductively coupled plasma-mass spectrometry (ICP-MS). Cadmium levels in blood were statistically significantly correlated with cadmium levels in kidneys. The cadmium uptake was not higher in iron-deficient suckling piglets; rather, we detected a higher cadmium uptake in the iron-supplemented ones. The expression and localisation of DMT1, FPN1 and MT were not affected by iron status and could therefore not explain the findings. Our results suggest that there are developmental differences in the handling of both iron and cadmium in newborns as compared to adults.

Orchestration of dynamic copper navigation - new and missing pieces.

A general principle in all cells in the body is that an essential metal - here copper - is taken up at the plasma membrane, directed through cellular compartments for use in specific enzymes and pathways, stored in specific scavenging molecules if in surplus, and finally expelled from the cells. Here we attempt to provide a critical view on key concepts involved in copper transfer across membranes and through compartments in the human body. The focus of this review is on the influence of bioinorganic and thermodynamic rules on the flow in cellular copper networks. Transition of copper from one oxidation state to another will often lead to errant electrons that are highly reactive and prone to form radicals and reactive oxygen or nitrogen species (ROS and RNS). Strict control of potentially toxic oxidative species is an important part of understanding the edge of human copper metabolism. The present review critically covers translocation across simple and complex membranes as well as extracellular and intracellular copper routing. We discuss in depth four tissues with polarized cell barriers - the gut, liver, kidneys, and brain - to illustrate the similarities and differences in transcellular transfer. Copper chaperoning, buffering and binding dynamics to guide the metal to different sites are also covered, while individual molecular interaction kinetics are not detailed. Sorting and targeting mechanisms and principles crucial for correct localisation will also be touched upon.

Acidic pH is essential for maintaining mast cell secretory granule homeostasis.

It has been recognized for a long time that the secretory granules of mast cells are acidic, but the functional importance of maintaining an acidic pH in the mast cell granules is not fully understood. Here we addressed this issue by examining the effects of raising the pH of the mast cell secretory granules. Mast cells were incubated with bafilomycin A1, an inhibitor of the vacuolar-type ATPase proton pump. Supporting a role of vacuolar-type ATPase in mast cell granule acidification, bafilomycin A1 treatment caused a robust increase in granule pH. This was accompanied by marked effects on mast cell granules, including swelling and acquisition of vacuole-like morphology. Moreover, bafilomycin A1 caused extensive, yet selective effects on the granule content. These included aberrant processing of pro-carboxypeptidase A3 and a reduction in the level of intracellular histamine, the latter being accompanied by an increase in extracellular histamine. In contrast, the storage of ß-hexosaminidase, a prototype lysosomal hydrolase known to be stored in mast cell granules, was not affected by abrogation of granule acidification. Moreover, bafilomycin A1 caused a reduction of tryptase enzymatic activity and appearance of tryptase degradation products. Tryptase inhibition prevented the formation of such degradation products, suggesting that the pH elevation causes tryptase to undergo autoproteolysis. Taken together, our findings reveal that mast cell secretory granule homeostasis is critically dependent on an acidic milieu.

The combined action of mast cell chymase, tryptase and carboxypeptidase A3 protects against melanoma colonization of the lung.

Mast cell secretory granules are densely packed with various bioactive mediators including proteases of chymase, tryptase and CPA3 type. Previous studies have indicated that mast cells can affect the outcome of melanoma but the contribution of the mast cell granule proteases to such effects has not been clear. Here we addressed this issue by assessing mice lacking either the chymase Mcpt4, the tryptase Mcpt6 or carboxypeptidase A3 (Cpa3), as well as mice simultaneously lacking all three proteases, in a model of melanoma dissemination from blood to the lung. Although mice with individual deficiency in the respective proteases did not differ significantly from wildtype mice in the extent of melanoma colonization, mice with multiple protease deficiency (Mcpt4/Mcpt6/Cpa3-deficient) exhibited a higher extent of melanoma colonization in lungs as compared to wildtype animals. This was supported by higher expression of melanoma-specific genes in lungs of Mcpt4/Mcpt6/CPA3-deficient vs. wildtype mice. Cytokine profiling showed that the levels of CXCL16, a chemokine with effects on T cell populations and NKT cells, were significantly lower in lungs of Mcpt4/Mcpt6/Cpa3-deficient animals vs. controls, suggesting that multiple mast cell protease deficiency might affect T cell or NKT cell populations. In line with this, we found that the Mcpt4/Mcpt6/Cpa3-deficiency was associated with a reduction in cells expressing CD1d, a MHC class 1-like molecule that is crucial for presenting antigen to invariant NKT (iNKT) cells. Together, these findings indicate a protective role of mast cell-specific proteases in melanoma dissemination, and suggest that this effect involves a CXCL16/CD1d/NKT cell axis.

Cadmium-induced disturbances in lactating mammary glands of mice.

Previous studies in various species have demonstrated that cadmium levels in milk are low and that cadmium is retained in the mammary glands of lactating rodents. The objective of the present study was to examine mammary glands following cadmium exposure during peak lactation. Mice were given 5 microg, 100 microg or 2000 microg cadmium/kg body weight subcutaneously on lactation days 8-10 and mammary glands were dissected on lactation day 11 for histology, quantitative gene expression and mineral analyses. Cadmium exposure induced morphological changes in the lactating mammary gland. A remodelling of the lactating mammary tissue including an increase in fat content, a less active feature of the mammary alveolar epithelial cells and more condensed appearance of the milk alveoli were observed. Although these changes were most prominent in the animals exposed to the highest cadmium dose similar morphological alterations were indicated at the two lower doses. A significant negative dose-response relationship between beta-casein gene expression and exposure of cadmium was demonstrated, while mRNA levels of alpha-lactalbumin were not affected by cadmium treatment. Furthermore, reduced levels of calcium in the mammary glands of the dams exposed to the highest cadmium dose and a positive correlation between calcium and beta-casein were demonstrated. No other differences were detected among the cadmium dose groups in mammary levels of calcium, zinc, iron or copper. In conclusion, our results indicate that cadmium disturbs the function of the lactating mammary gland, which consequently may impair the development of the suckling offspring.

Mast cells promote melanoma colonization of lungs.

Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre- R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre- R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre- R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

Reduced thyroxine levels in mice perinatally exposed to polybrominated diphenyl ethers.

The aim of the study was to follow plasma thyroxine levels and hepatic enzyme activities in offspring after maternal gestational and lactational exposure to polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls. Mice were given 10 equimolar oral doses from gestational day (GD) 4 to postnatal day (PND) 17 of either Bromkal 70-5DE, 2,2',4,4',5-pentabrominated diphenyl ether (BDE-99) or Aroclor 1254 (total dose of 0.80mmol/kg, b.w.). Plasma thyroxine levels were reduced in offspring in the Aroclor and Bromkal groups on PND11 but had returned to control levels by PND37. No effects on thyroxine levels were seen in the dams. Hepatic activity of EROD was increased in all treated offspring groups and so was UDP-GT in Aroclor-exposed offspring on PND11 and PND18. This study shows that PBDEs and PCBs, probably after microsomal transformation, have endocrine disrupting properties in perinatally exposed juvenile mice, most pronounced at PND11. However, BDE-99 had no effect on thyroxine levels, suggesting that other components in Bromkal are responsible for the hypothyroxinemia.

The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy.

Copper (Cu) is an essential metal for growth and development that has the potential to be toxic if levels accumulate beyond the ability of cells to homeostatically balance uptake with detoxification. One system for Cu acquisition is the integral membrane Cu(+) transporter, Ctr1, which has been quite well characterized in terms of its function and physiology. The mammalian Ctr2 protein has been a conundrum for the copper field, as it is structurally closely related to the high affinity Cu transporter Ctr1, sharing important motifs for Cu transport activity. However, in contrast to mammalian Ctr1, Ctr2 fails to suppress the Cu-dependent growth phenotype of yeast cells defective in Cu(+) import, nor does it appreciably stimulate Cu acquisition when over-expressed in mammalian cells, underscoring important functional dissimilarities between the two proteins. Several roles for the mammalian Ctr2 have been suggested both in vitro and in vivo. Here, we summarize and discuss current insights into the Ctr2 protein and its interaction with Ctr1, its functions in mammalian Cu homeostasis and platinum-based chemotherapy.

How copper traverses cellular membranes through the mammalian copper transporter 1, Ctr1.

The copper transporter 1, Ctr1, is part of a major pathway for cellular copper (Cu) uptake in the intestinal epithelium, in hepatic and cardiac tissue, and likely in many other mammalian cells and tissues. Here, we summarize what is currently known about how extracellular Cu travels across the plasma membrane to enter the cytoplasm for intracellular distribution and for use by proteins and enzymes, the physiological roles of Ctr1, and its regulation. As a critical Cu importer, Ctr1 occupies a strategic position to exert a strong modifying influence on diseases and pathophysiological states caused by imbalances in Cu homeostasis. A more thorough understanding of the mechanisms that regulate Ctr1 abundance, trafficking, and function will provide new insights and opportunities for disease therapies.

Mefloquine, an anti-malaria agent, causes reactive oxygen species-dependent cell death in mast cells via a secretory granule-mediated pathway.

Mast cells are known to have a detrimental impact on a variety of pathological conditions. There is therefore an urgent need of developing strategies that limit their harmful effects. The aim of this study was to accomplish this by developing a means of inducing mast cell apoptosis. The strategy was to identify novel compounds that induce mast cell apoptosis by permeabilization of their secretory lysosomes (granules). As a candidate, we assessed mefloquine, an anti-malarial drug that has been proposed to have lysosome-permeabilizing activity. Mefloquine was added to mast cells and administered in vivo, followed by assessment of the extent and mechanisms of mast cell death. Mefloquine was cytotoxic to murine and human mast cells. Mefloquine induced apoptotic cell death of wild-type mast cells whereas cells lacking the granule compounds serglycin proteoglycan or tryptase were shown to undergo necrotic cell death, the latter finding indicating a role of the mast cell granules in mefloquine-induced cell death. In support of this, mefloquine was shown to cause compromised granule integrity and to induce leakage of granule components into the cytosol. Mefloquine-induced cell death was refractory to caspase inhibitors but was completely abrogated by reactive oxygen species inhibition. These findings identify mefloquine as a novel anti-mast cell agent, which induces mast cell death through a granule-mediated pathway. Mefloquine may thus become useful in therapy aiming at limiting harmful effects of mast cells.