The biology of mammalian multi-copper ferroxidases.

The mammalian multicopper ferroxidases (MCFs) ceruloplasmin (CP), hephaestin (HEPH) and zyklopen (ZP) comprise a family of conserved enzymes that are essential for body iron homeostasis. Each of these enzymes contains six biosynthetically incorporated copper atoms which act as intermediate electron acceptors, and the oxidation of iron is associated with the four electron reduction of dioxygen to generate two water molecules. CP occurs in both a secreted and GPI-linked (membrane-bound) form, while HEPH and ZP each contain a single C-terminal transmembrane domain. These enzymes function to ensure the efficient oxidation of iron so that it can be effectively released from tissues via the iron export protein ferroportin and subsequently bound to the iron carrier protein transferrin in the blood. CP is particularly important in facilitating iron release from the liver and central nervous system, HEPH is the major MCF in the small intestine and is critical for dietary iron absorption, and ZP is important for normal hair development. CP and HEPH (and possibly ZP) function in multiple tissues. These proteins also play other (non-iron-related) physiological roles, but many of these are ill-defined. In addition to disrupting iron homeostasis, MCF dysfunction perturbs neurological and immune function, alters cancer susceptibility, and causes hair loss, but, despite their importance, how MCFs co-ordinately maintain body iron homeostasis and perform other functions remains incompletely understood.

Biallelic HEPHL1 variants impair ferroxidase activity and cause an abnormal hair phenotype.

Maintenance of the correct redox status of iron is functionally important for critical biological processes. Multicopper ferroxidases play an important role in oxidizing ferrous iron, released from the cells, into ferric iron, which is subsequently distributed by transferrin. Two well-characterized ferroxidases, ceruloplasmin (CP) and hephaestin (HEPH) facilitate this reaction in different tissues. Recently, a novel ferroxidase, Hephaestin like 1 (HEPHL1), also known as zyklopen, was identified. Here we report a child with compound heterozygous mutations in HEPHL1 (NM_001098672) who presented with abnormal hair (pili torti and trichorrhexis nodosa) and cognitive dysfunction. The maternal missense mutation affected mRNA splicing, leading to skipping of exon 5 and causing an in-frame deletion of 85 amino acids (c.809_1063del; p.Leu271_ala355del). The paternal mutation (c.3176T>C; p.Met1059Thr) changed a highly conserved methionine that is part of a typical type I copper binding site in HEPHL1. We demonstrated that HEPHL1 has ferroxidase activity and that the patient's two mutations exhibited loss of this ferroxidase activity. Consistent with these findings, the patient's fibroblasts accumulated intracellular iron and exhibited reduced activity of the copper-dependent enzyme, lysyl oxidase. These results suggest that the patient's biallelic variants are loss-of-function mutations. Hence, we generated a Hephl1 knockout mouse model that was viable and had curly whiskers, consistent with the hair phenotype in our patient. These results enhance our understanding of the function of HEPHL1 and implicate altered ferroxidase activity in hair growth and hair disorders.

The Placental Ferroxidase Zyklopen Is Not Essential for Iron Transport to the Fetus in Mice.

BACKGROUND: The ferroxidase zyklopen (Zp) has been implicated in the placental transfer of iron to the fetus. However, the evidence for this is largely circumstantial. OBJECTIVES: This study aimed to determine whether Zp is essential for placental iron transfer. METHODS: A model was established using 8- to 12-wk-old pregnant C57BL/6 mice on standard rodent chow in which Zp was knocked out in the fetus and fetal components of the placenta. Zp was also disrupted in the entire placenta using global Zp knockout mice. Inductively coupled plasma MS was used to measure total fetal iron, an indicator of the amount of iron transferred by the placenta to the fetus, at embryonic day 18.5 of gestation. Iron transporter expression in the placenta was measured by Western blotting, and the expression of Hamp1, the gene encoding the iron regulatory hormone hepcidin, was determined in fetal liver by real-time PCR. RESULTS: There was no change in the amount of iron transferred to the fetus when Zp was disrupted in either the fetal component of the placenta or the entire placenta. No compensatory changes in the expression of the iron transport proteins transferrin receptor 1 or ferroportin were observed, nor was there any change in fetal liver Hamp1 mRNA. Hephl1, the gene encoding Zp, was expressed mainly in the maternal decidua of the placenta and not in the nutrient-transporting syncytiotrophoblast. Disruption of Zp in the whole placenta resulted in a 26% increase in placental size (P < 0.01). CONCLUSIONS: Our data indicate that Zp is not essential for the efficient transfer of iron to the fetus in mice and is localized predominantly in the maternal decidua. The increase in placental size observed when Zp is knocked out in the entire placenta suggests that this protein may play a role in placental development.

Genetic screens reveal CCDC115 as a modulator of erythroid iron and heme trafficking.

Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells.

Multiple environmental stressors induce complex transcriptomic responses indicative of phenotypic outcomes in Western fence lizard.

BACKGROUND: The health and resilience of species in natural environments is increasingly challenged by complex anthropogenic stressor combinations including climate change, habitat encroachment, and chemical contamination. To better understand impacts of these stressors we examined the individual- and combined-stressor impacts of malaria infection, food limitation, and 2,4,6-trinitrotoluene (TNT) exposures on gene expression in livers of Western fence lizards (WFL, Sceloporus occidentalis) using custom WFL transcriptome-based microarrays. RESULTS: Computational analysis including annotation enrichment and correlation analysis identified putative functional mechanisms linking transcript expression and toxicological phenotypes. TNT exposure increased transcript expression for genes involved in erythropoiesis, potentially in response to TNT-induced anemia and/or methemoglobinemia and caused dose-specific effects on genes involved in lipid and overall energy metabolism consistent with a hormesis response of growth stimulation at low doses and adverse decreases in lizard growth at high doses. Functional enrichment results were indicative of inhibited potential for lipid mobilization and catabolism in TNT exposures which corresponded with increased inguinal fat weights and was suggestive of a decreased overall energy budget. Malaria infection elicited enriched expression of multiple immune-related functions likely corresponding to increased white blood cell (WBC) counts. Food limitation alone enriched functions related to cellular energy production and decreased expression of immune responses consistent with a decrease in WBC levels. CONCLUSIONS: Despite these findings, the lizards demonstrated immune resilience to malaria infection under food limitation with transcriptional results indicating a fully competent immune response to malaria, even under bio-energetic constraints. Interestingly, both TNT and malaria individually increased transcriptional expression of immune-related genes and increased overall WBC concentrations in blood; responses that were retained in the TNT x malaria combined exposure. The results demonstrate complex and sometimes unexpected responses to multiple stressors where the lizards displayed remarkable resiliency to the stressor combinations investigated.

Effects of dietary iron deficiency or overload on bone: Dietary details matter.

PURPOSE: Bone is susceptible to fluctuations in iron homeostasis, as both iron deficiency and overload are linked to poor bone strength in humans. In mice, however, inconsistent results have been reported, likely due to different diet setups or genetic backgrounds. Here, we assessed the effect of different high and low iron diets on bone in six inbred mouse strains (C57BL/6J, A/J, BALB/cJ, AKR/J, C3H/HeJ, and DBA/2J). METHODS: Mice received a high (20,000 ppm) or low-iron diet (∼10 ppm) after weaning for 6-8 weeks. For C57BL/6J males, we used two dietary setups with similar amounts of iron, yet different nutritional compositions that were either richer ("TUD study") or poorer ("UCLA study") in minerals and vitamins. After sacrifice, liver, blood and bone parameters as well as bone turnover markers in the serum were analyzed. RESULTS: Almost all mice on the UCLA study high iron diet had a significant decrease of cortical and trabecular bone mass accompanied by high bone resorption. Iron deficiency did not change bone microarchitecture or turnover in C57BL/6J, A/J, and DBA/2J mice, but increased trabecular bone mass in BALB/cJ, C3H/HeJ and AKR/J mice. In contrast to the UCLA study, male C57BL/6J mice in the TUD study did not display any changes in trabecular bone mass or turnover on high or low iron diet. However, cortical bone parameters were also decreased in TUD mice on the high iron diet. CONCLUSION: Thus, these data show that cortical bone is more susceptible to iron overload than trabecular bone and highlight the importance of a nutrient-rich diet to potentially mitigate the negative effects of iron overload on bone.

Genome-wide CRISPR/Cas9 screen identifies etoposide response modulators associated with clinical outcomes in pediatric AML.

Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.

Overcoming Gemcitabine Resistance in Pancreatic Cancer Using the BCL-XL-Specific Degrader DT2216.

Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.

CRISPR Screens in Toxicology Research: An Overview.

The use of genome editing tools is expanding our understanding of various human diseases by providing insight into gene-disease interactions. Despite the recognized role of toxicants in the development of human health issues and conditions, there is currently limited characterization of their mechanisms of action, and the application of CRISPR-based genome editing to the study of toxicants could help in the identification of novel gene-environment interactions. CRISPR-based functional screens enable identification of cellular mechanisms fundamental for response and susceptibility to a given toxicant. The aim of this review is to inform future directions in the application of CRISPR technologies in toxicological studies. We review and compare different types of CRISPR-based methods including pooled, anchored, combinatorial, and perturb-sequencing screens in vitro, in addition to pooled screenings in model organisms. © 2021 Wiley Periodicals LLC.

Transcriptomic response patterns of hornyhead turbot (Pleuronichthys verticalis) dosed with polychlorinated biphenyls and polybrominated diphenyl ethers.

To evaluate the impact of environmental contaminants on aquatic health, extensive surveys of fish populations have been conducted using bioaccumulation as an indicator of impairment. While these studies have reported mixtures of chemicals in fish tissues, the relationship between specific contaminants and observed adverse impacts remains poorly understood. The present study aimed to characterize the toxicological responses induced by persistent organic pollutants in wild-caught hornyhead turbot (P. verticalis). To do so, hornyhead turbot were interperitoneally injected with a single dose of PCB or PBDE congeners prepared using environmentally realistic mixture proportions. After 96-hour exposure, the livers were excised and analyzed using transcriptomic approaches and analytical chemistry. Concentrations of PCBs and PBDEs measured in the livers indicated clear differences across treatments, and congener profiles closely mirrored our expectations. Distinct gene profiles were characterized for PCB and PBDE exposed fish, with significant differences observed in the expression of genes associated with immune responses, endocrine-related functions, and lipid metabolism. Our findings highlight the key role that transcriptomics can play in monitoring programs to assess chemical-induced toxicity in heterogeneous group of fish (mixed gender and life stage) as is typically found during field surveys. Altogether, the present study provides further evidence of the potential of transcriptomic tools to improve aquatic health assessment and identify causative agents.

Treatment with HIV-Protease Inhibitor Nelfinavir Identifies Membrane Lipid Composition and Fluidity as a Therapeutic Target in Advanced Multiple Myeloma.

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)-refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9-based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI-refractory multiple myeloma. SIGNIFICANCE: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.

Mammalian iron transport.

Iron is essential for basic cellular processes but is toxic when present in excess. Consequently, iron transport into and out of cells is tightly regulated. Most iron is delivered to cells bound to plasma transferrin via a process that involves transferrin receptor 1, divalent metal-ion transporter 1 and several other proteins. Non-transferrin-bound iron can also be taken up efficiently by cells, although the mechanism is poorly understood. Cells can divest themselves of iron via the iron export protein ferroportin in conjunction with an iron oxidase. The linking of an oxidoreductase to a membrane permease is a common theme in membrane iron transport. At the systemic level, iron transport is regulated by the liver-derived peptide hepcidin which acts on ferroportin to control iron release to the plasma.

Linking mechanistic and behavioral responses to sublethal esfenvalerate exposure in the endangered delta smelt; Hypomesus transpacificus (Fam. Osmeridae).

BACKGROUND: The delta smelt (Hypomesus transpacificus) is a pelagic fish species listed as endangered under both the USA Federal and Californian State Endangered Species Acts and considered an indicator of ecosystem health in its habitat range, which is limited to the Sacramento-San Joaquin estuary in California, USA. Anthropogenic contaminants are one of multiple stressors affecting this system, and among them, current-use insecticides are of major concern. Interrogative tools are required to successfully monitor effects of contaminants on the delta smelt, and to research potential causes of population decline in this species. We have created a microarray to investigate genome-wide effects of potentially causative stressors, and applied this tool to assess effects of the pyrethroid insecticide esfenvalerate on larval delta smelt. Selected genes were further investigated as molecular biomarkers using quantitative PCR analyses. RESULTS: Exposure to esfenvalerate affected swimming behavior of larval delta smelt at concentrations as low as 0.0625 mug.L-1, and significant differences in expression were measured in genes involved in neuromuscular activity. Alterations in the expression of genes associated with immune responses, along with apoptosis, redox, osmotic stress, detoxification, and growth and development appear to have been invoked by esfenvalerate exposure. Swimming impairment correlated significantly with expression of aspartoacylase (ASPA), an enzyme involved in brain cell function and associated with numerous human diseases. Selected genes were investigated for their use as molecular biomarkers, and strong links were determined between measured downregulation in ASPA and observed behavioral responses in fish exposed to environmentally relevant pyrethroid concentrations. CONCLUSIONS: The results of this study show that microarray technology is a useful approach in screening for, and generation of molecular biomarkers in endangered, non-model organisms, identifying specific genes that can be directly linked with sublethal toxicological endpoints; such as changes in expression levels of neuromuscular genes resulting in measurable swimming impairments. The developed microarrays were successfully applied on larval fish exposed to esfenvalerate, a known contaminant of the Sacramento-San Joaquin estuary, and has permitted the identification of specific biomarkers which could provide insight into the factors contributing to delta smelt population decline.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

Multi-copper ferroxidase deficiency leads to iron accumulation and oxidative damage in astrocytes and oligodendrocytes.

Accumulation of iron has been associated with the pathobiology of various disorders of the central nervous system. Our previous work has shown that hephaestin (Heph) and ceruloplasmin (Cp) double knockout (KO) mice induced iron accumulation in multiple brain regions and that this was paralleled by increased oxidative damage and deficits in cognition and memory. In this study, we enriched astrocytes and oligodendrocytes from the cerebral cortex of neonatal wild-type (WT), Heph KO and Cp KO mice. We demonstrated that Heph is highly expressed in oligodendrocytes, while Cp is mainly expressed in astrocytes. Iron efflux was impaired in Cp KO astrocytes and Heph KO oligodendrocytes and was associated with increased oxidative stress. The expression of Heph, Cp, and other iron-related genes was examined in astrocytes and oligodendrocytes both with and without iron treatment. Interestingly, we found that the expression of the mRNA encoding ferroportin 1, a transmembrane protein that cooperates with CP and HEPH to export iron from cells, was positively correlated with Cp expression in astrocytes, and with Heph expression in oligodendrocytes. Our findings collectively demonstrate that HEPH and CP are important for the prevention of glial iron accumulation and thus may be protective against oxidative damage.

Déjà vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins.

After reading many 2-DE-based articles featuring lists of the differentially expressed proteins, one starts experiencing a disturbing déjà vu. The same proteins seem to predominate regardless of the experiment, tissue or species. To quantify the occurrence of individual differentially expressed proteins in 2-DE experiment reports, we compiled the identities of differentially expressed proteins identified in human, mouse, and rat tissues published in three recent volumes of Proteomics and calculated the appearance of the most predominant proteins in the dataset. The most frequently identified protein is a highly abundant glycolytic enzyme enolase 1, differentially expressed in nearly every third experiment on both human and rodent tissues. Heat-shock protein 27 (HSP27) and heat-shock protein 60 (HSP60) were differentially expressed in about 30 percent of human and rodent samples, respectively. Considering protein families as units, keratins and peroxiredoxins are the most frequently identified molecules, with at least one member of the group being differentially expressed in about 40 percent of all experiments. We suggest that the frequent identification of these proteins must be considered in the interpretation of any 2-DE studies. We consider if these commonly observed changes represent common cellular stress responses or are a reflection of the technical limitations of 2-DE.

Iron-independent specific protein expression pattern in the liver of HFE-deficient mice.

Hereditary hemochromatosis type I is an autosomal-recessive iron overload disease associated with a mutation in HFE gene. The most common mutation, C282Y, disrupts the disulfide bond necessary for the association of HFE with beta-2-microglobulin and abrogates cell surface HFE expression. HFE-deficient mice develop iron overload indicating a central role of the protein in the pathogenesis of hereditary hemochromatosis type I. However, despite significant effort, the role of the HFE protein in iron metabolism is still unknown. To shed a light on the molecular mechanism of HFE-related hemochromatosis we studied protein expression changes elicited by HFE-deficiency in the liver which is the organ critical for the regulation of iron metabolism. We undertook a proteomic study comparing protein expression in the liver of HFE deficient mice with control animals. We compared HFE-deficient animals with control animals with identical iron levels obtained by dietary treatment to identify changes specific to HFE deficiency rather than iron loading. We found 11 proteins that were differentially expressed in the HFE-deficient liver using two-dimensional electrophoresis and mass spectrometry identification. Of particular interest were urinary proteins 1, 2 and 6, glutathione-S-transferase P1, selenium binding protein 2, sarcosine dehydrogenase and thioredoxin-like protein 2. Our data suggest possible involvement of lipocalins, TNF-alpha signaling and PPAR alpha regulatory pathway in the pathogenesis of hereditary hemochromatosis and suggest future targeted research addressing the roles of the identified candidate genes in the molecular mechanism of hereditary hemochromatosis.

Effects of strain and age on hepatic gene expression profiles in murine models of HFE-associated hereditary hemochromatosis.

Hereditary hemochromatosis is an iron overload disorder most commonly caused by a defect in the HFE gene. While the genetic defect is highly prevalent, the majority of individuals do not develop clinically significant iron overload, suggesting the importance of genetic modifiers. Murine hfe knockout models have demonstrated that strain background has a strong effect on the severity of iron loading. We noted that hepatic iron loading in hfe-/- mice occurs primarily over the first postnatal weeks (loading phase) followed by a timeframe of relatively static iron concentrations (plateau phase). We thus evaluated the effects of background strain and of age on hepatic gene expression in Hfe knockout mice (hfe-/-). Hepatic gene expression profiles were examined using cDNA microarrays in 4- and 8-week-old hfe-/- and wild-type mice on two different genetic backgrounds, C57BL/6J (C57) and AKR/J (AKR). Genes differentially regulated in all hfe-/- mice groups, compared with wild-type mice, including those involved in cell survival, stress and damage responses and lipid metabolism. AKR strain-specific changes in lipid metabolism genes and C57 strain-specific changes in cell adhesion and extracellular matrix protein genes were detected in hfe-/- mice. Mouse strain and age are each significantly associated with hepatic gene expression profiles in hfe-/- mice. These affects may underlie or reflect differences in iron loading in these mice.

Iron deficiency upregulates Egr1 expression.

Iron-deficient anemia is a prevalent disease among humans. We searched for genes regulated by iron deficiency and its regulated mechanism. cDNA microarrays were performed using Hepa1c1c7 cells treated with 100 µM desferrioxamine (DFO), an iron chelator. Early growth response 1 (Egr1) was upregulated with at least 20-fold increase within 4 h and lasted for 24 h, which was confirmed by qRT-PCR. This activation was not seen by ferric ammonium citrate (FAC). DFO increased the transcriptional activity of Egr1-luc (-604 to +160) and serum response element (SRE)-luc reporters by 2.7-folds. In addition, cycloheximide lowered DFO-induced Egr1 mRNA levels. The upregulation of Egr1 by DFO was accompanied by sustained ERK signals along with phosphorylation of Elk-1. The ERK inhibitor (PD98059) prevented the DFO-induced Egr1 mRNAs. Overexpression of Elk-1 mutant (pElk-1S383A) decreased Egr1 reporter activity. DFO lowered reactive oxygen species (ROS) production and increased caspase 3/7 activity and cell death. DFO-induced iron deficiency upregulates Egr1 in part through transcriptional activation via ERK and Elk-1 signals, which may be important in the regulation of cell death in hepatoma cells. Our study demonstrated that iron depletion controlled the expression of Egr1, which might contribute to decisions about cellular fate in response to iron deficiency.

Gene expression profiling in chronic copper overload reveals upregulation of Prnp and App.

The level at which copper becomes toxic is not clear. Several studies have indicated that copper causes oxidative stress; however, most have tested very high levels of copper exposure. We currently have only a limited understanding of the protective systems that operate in cells chronically exposed to copper. Additionally, the limits of homeostatic regulation are not known, making it difficult to define the milder effects of copper excess. Furthermore, a robust assay to facilitate the diagnosis of copper excess and to distinguish mild, moderate, and severe copper overload is needed. To address these issues, we have investigated the effects on steady-state gene expression of chronic copper overload in a cell culture model system using cDNA microarrays. For this study we utilized cells from genetic models of copper overload: fibroblast cells from two mouse mutants, C57BL/6-Atp7a(Mobr) and C57BL/6-Atp7a(Modap). These cell lines accumulate copper to abnormally high levels in normal culture media due to a defect in copper export from the cell. We identified 12 differentially expressed genes in common using our outlier identification methods. Surprisingly, our results show no evidence of oxidative stress in the copper-loaded cells. In addition, candidate components perhaps responsible for a copper-specific homeostatic response are identified. The genes that encode for the prion protein and the amyloid-beta precursor protein, two known copper-binding proteins, are upregulated in both cell lines.