Gene-Environment Interactions: My Unique Journey.

I am deeply honored to be invited to write this scientific autobiography. As a physician-scientist, pediatrician, molecular biologist, and geneticist, I have authored/coauthored more than 600 publications in the fields of clinical medicine, biochemistry, biophysics, pharmacology, drug metabolism, toxicology, molecular biology, cancer, standardized gene nomenclature, developmental toxicology and teratogenesis, mouse genetics, human genetics, and evolutionary genomics. Looking back, I think my career can be divided into four distinct research areas, which I summarize mostly chronologically in this article: (a) discovery and characterization of the AHR/CYP1 axis, (b) pharmacogenomics and genetic prediction of response to drugs and other environmental toxicants, (c) standardized drug-metabolizing gene nomenclature based on evolutionary divergence, and (d) discovery and characterization of the SLC39A8 gene encoding the ZIP8 metal cation influx transporter. Collectively, all four topics embrace gene-environment interactions, hence the title of my autobiography.

Hepatobiliary manganese homeostasis is dynamic in the setting of inflammation or infection in mice.

Manganese is a diet-derived micronutrient that is essential for critical cellular processes like redox homeostasis, protein glycosylation, and lipid and carbohydrate metabolism. Control of Mn availability, especially at the local site of infection, is a key component of the innate immune response. Less has been elucidated about Mn homeostasis at the systemic level. In this work, we demonstrate that systemic Mn homeostasis is dynamic in response to inflammation and infection in mice. This phenomenon is evidenced in male and female mice, mice of two genetic backgrounds (C57BL/6 and BALB/c), in multiple models of acute (dextran sodium sulfate-induced) and chronic (enterotoxigenic Bacteroides fragilis) colitis, and systemic infection with Candida albicans. When mice were fed a standard corn-based chow with excess Mn (100 ppm), liver Mn decreased and biliary Mn increased threefold in response to infection or colitis. Liver iron, copper, and zinc were unchanged. When dietary Mn was restricted to minimally adequate amounts (10 ppm), baseline hepatic Mn levels decreased by approximately 60% in the liver, and upon induction of colitis, liver Mn did not decrease further, however, biliary Mn still increased 20-fold. In response to acute colitis, hepatic Slc39a8 mRNA (gene encoding the Mn importer, Zip8) and Slc30a10 mRNA (gene encoding the Mn exporter, Znt10) are decreased. Zip8 protein is decreased. Inflammation/infection-associated dynamic Mn homeostasis may represent a novel host immune/inflammatory response that reorganizes systemic Mn availability through differential expression of key Mn transporters with down-regulation of Zip8.

Reduced Mn uptake of pleiotropic ZIP8 SNP is caused by its loss of Mn-responsive accumulation on the cell-surface.

Zrt/Irt-like protein 8 (ZIP8), which is a Zn transporter, plays a pivotal role as a Mn transporter. Recent studies have shown that a ZIP8 SNP (rs13107325 C→T, A391T) is associated with multiple diseases, likely by causing systemic Mn deficiency. However, the underlying molecular mechanisms remain unclear. We attempted to address this issue in cell-based experiments using Madin-Darby canine kidney cells stably expressing ZIP8 WT or the A391T SNP mutant under the control of the Tet-regulatable promoter. We showed that the A391T mutant lost the property of Mn-responsive accumulation on the cell surface, which was observed in WT ZIP8. We also showed that the loss of Mn-responsive accumulation of A391T mutant was associated with its reduced Mn uptake, compared with WT ZIP8, in the Mn uptake assay using the radioisotope 54Mn. Our results potentially explain how the ZIP8 A391T substitution is associated with disease pathogenesis caused by Mn deficiency.

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells.

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn2+, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca2+-uptake transporter thought to support Golgi uptake of Mn2+ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca2+ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca2+ levels. In turn, we found increased cytoplasmic Ca2+ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in 54Mn2+ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca2+ and 54Mn2+ accumulation. While addition of Ca2+ positively regulated ZIP-mediated 54Mn2+ uptake, we show chelation of Ca2+ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn2+ and other divalent metal ions-mediated ZIP metal transporters.

The Regulation of ZIP8 by Dietary Manganese in Mice.

ZIP8 is a newly identified manganese transporter. A lack of functional ZIP8 results in severe manganese deficiency in both humans and mice, indicating that ZIP8 plays a crucial role in maintaining body manganese homeostasis. Despite a well-acknowledged connection between ZIP8 and manganese metabolism, how ZIP8 is regulated under high-manganese conditions remains unclear. The primary goal of this study was to examine the regulation of ZIP8 by high-manganese intake. We used both neonatal and adult mouse models in which mice were supplied with dietary sources containing either a normal or a high level of manganese. We discovered that high-manganese intake caused a reduction in liver ZIP8 protein in young mice. Since a decrease in hepatic ZIP8 leads to reduced manganese reabsorption from the bile, our study identified a novel mechanism for the regulation of manganese homeostasis under high-manganese conditions: high dietary manganese intake results in a decrease in ZIP8 in the liver, which in turn decreases the reabsorption of manganese from the bile to prevent manganese overload in the liver. Interestingly, we found that a high-manganese diet did not cause a decrease in hepatic ZIP8 in adult animals. To determine the potential reason for this age-dependent variation, we compared the expressions of liver ZIP8 in 3-week-old and 12-week-old mice. We found that liver ZIP8 protein content in 12-week-old mice decreases when compared with that of 3-week-old mice under normal conditions. Overall, results from this study provide novel insights to facilitate the understanding of ZIP8's function in regulating manganese metabolism.

Proton-Pump Inhibitors Suppress T Cell Response by Shifting Intracellular Zinc Distribution.

Proton-pump inhibitors (PPI), e.g., omeprazole or pantoprazole, are the most widely used drugs for various gastrointestinal diseases. However, more and more side effects, especially an increased risk of infections, have been reported in recent years. The underlying mechanism has still not yet been fully uncovered. Hence, in this study, we analyzed the T cell response after treatment with pantoprazole in vitro. Pantoprazole preincubation reduced the production and secretion of interferon (IFN)-γ and interleukin (IL)-2 after the T cells were activated with phytohemagglutinin (PHA)-L or toxic shock syndrome toxin-1 (TSST-1). Moreover, a lower zinc concentration in the cytoplasm and a higher concentration in the lysosomes were observed in the pantoprazole-treated group compared to the untreated group. We also tested the expression of the zinc transporter Zrt- and Irt-like protein (Zip)8, which is located in the lysosomal membrane and plays a key role in regulating intracellular zinc distribution after T cell activation. Pantoprazole reduced the expression of Zip8. Furthermore, we measured the expression of cAMP-responsive element modulator (CREM) α, which directly suppresses the expression of IL-2, and the expression of the phosphorylated cAMP response element-binding protein (pCREB), which can promote the expression of IFN-γ. The expression of CREMα was dramatically increased, and different isoforms appeared, whereas the expression of pCREB was downregulated after the T cells were treated with pantoprazole. In conclusion, pantoprazole downregulates IFN-γ and IL-2 expression by regulating the expression of Zip8 and pCREB or CREMα, respectively.

Effects of individual amino acid mutations of zinc transporter ZIP8 on manganese- and cadmium-transporting activity.

Zinc (Zn) transporter ZIP8, encoded by SLC39A8, is a unique transporter that can transport divalent manganese (Mn) and cadmium (Cd) in addition to Zn. Recently, associations between various human diseases and variant forms of ZIP8 have been reported. Four amino acid residues, V33, G38, S335, and I340, of human ZIP8 (hZIP8) are mutated in patients with congenital disorders of glycosylation (CDG), whose blood Mn levels are extremely low. Many genome-wide association studies have reported that the A391T mutation of hZIP8 caused by rs13107325 is associated with a wide range of diseases. However, the roles of individual mutations of hZIP8 on metal-transporting activity remain elusive. We established DT40 cells respectively expressing the four mutant hZIP8s and compared the Mn- and Cd-transporting activity between the mutants and wild-type hZIP8. Among the four mutations observed in the ZIP8-mutated CDG patients, the S335T and I340 N mutations in the predicted transmembrane domain 5 (TMD5) completely abolished Mn- and Cd-transporting activity, while V33 M or G35R mutations at the N-terminus did not. We also examined the A391T mutation, which slightly reduced metal transporting activity. Finally, we examined the effects of artificial mutations in the metal-binding motif EEXXH in the TMD5. Replacing EEXXH with HEXXH, which exists in most ZIP transporters, abolished the Mn- and Cd-transporting activity of hZIP8, indicating that glutamic acid in this motif plays a critical role in the unique affinity of ZIP8 for Mn and Cd. Thus, the utilization of DT40 cells enabled us to clarify the different functions of each residue of hZIP8 on metal transport.

Dengue virus replication enhances labile zinc pools by modulation of ZIP8.

Zinc-dependent viral proteins rely on intracellular zinc homeostasis for successful completion of infectious life-cycle. Here, we report that the intracellular labile zinc levels were elevated at early stages of dengue virus (DENV) infection in hepatic cells and this increase in free zinc was abolished in cells infected with UV-inactivated virus or with a DENV replication inhibitor implicating a role for zinc homeostasis in viral RNA replication. This change in free zinc was mediated by zinc transporter, ZIP8, as siRNA-mediated knockdown of ZIP8 resulted in abrogation of increase in free zinc levels leading to significant reduction in DENV titers suggesting a crucial role for ZIP8 in early stages of DENV replication. Furthermore, elevated free zinc levels correlated with high copy numbers of dengue genome in peripheral blood leukocytes obtained from dengue patients compared to healthy controls suggesting a critical role for zinc homeostasis in dengue infection. TAKE AWAYS: Dengue virus utilises cellular zinc homeostasis during replication of its RNA. ZIP8 upregulates free zinc levels during dengue virus replication. Enhanced viremia associates with elevated intracellular free zinc in dengue.

ZIP8 mediates the extracellular matrix degradation of nucleus pulposus cells via NF-κB signaling pathway.

The extracellular matrix (ECM) degradation of nucleus pulposus cells (NPCs) is mainly induced by metalloproteinases (MMPs). Zn2+ is an essential component of MMPs, but the effect of Zn2+ importers in controlling ECM metabolism remains unclear. The purpose of this research was to identify the involvement of Zn2+ importers in ECM degradation induced by inflammatory stimuli and excessive mechanical stressing. In this study, NPCs from Sprague-Dawley (SD) rats were separated and cultured. FluoZin-3 AM staining was applied to detect [Zn2+]i in NPCs treated with Interleukin-1ß (IL-1ß) or cyclic tensile strain (CTS) with a Flexcell Strain Unit. We found that intracellular Zn2+ concentration ([Zn2+]i) elevated dramatically, and ZIP8 is the predominant Zn2+ importer among all importers in senescent NPCs. The [Zn2+]i and MMP expression level both increased in IL-1ß and CTS treated NPCs. Furthermore, the expression of ZIP8 was also markedly increased. However, knockdown of ZIP8 with siRNA alleviated ECM degradation induced by inflammatory stimuli and CTS. Both stimuli activated NF-κB signaling pathway, and knockdown of ZIP8 effectively inhibited NF-κB signaling pathway activation. In conclusion, knockdown of ZIP8 can alleviate NPCs' ECM degradation caused by inflammatory stimuli and excessive mechanical stressing.

Gestational diabetes mellitus affects placental iron homeostasis: Mechanism and clinical implications.

Clinical studies suggest that pregnant women with elevated iron levels are more vulnerable to develop gestational diabetes mellitus (GDM), but the causes and underlying mechanisms are unknown. We hypothesized that hyperglycemia induces cellular stress responses leading to dysregulated placental iron homeostasis. Hence, we compared the expression of genes/proteins involved in iron homeostasis in placentae from GDM and healthy pregnancies (n = 11 each). RT-qPCR and LC-MS/MS analyses revealed differential regulation of iron transporters/receptors (DMT1/FPN1/ZIP8/TfR1), iron sensors (IRP1), iron regulators (HEPC), and iron oxidoreductases (HEPH/Zp). To identify the underlying mechanisms, we adapted BeWo trophoblast cells to normoglycemic (N), hyperglycemic (H), and hyperglycemic-hyperlipidemic (HL) conditions and assessed Fe3+ -uptake, expression patterns, and cellular pathways involving oxidative stress (OS), ER-stress, and autophagy. H and HL induced alterations in cellular morphology, differential iron transporter expression, and reduced Fe3+ -uptake confirming the impact of hyperglycemia on iron transport observed in GDM patients. Pathway analysis and rescue experiments indicated that dysregulated OS and disturbed autophagy processes contribute to the reduced placental iron transport under hyperglycemic conditions. These adaptations could represent a protective mechanism preventing the oxidative damage for both fetus and placenta caused by highly oxidative iron. In pregnancies with risk for GDM, antioxidant treatment, and controlled iron supplementation could help to balance placental OS levels protecting mother and fetus from impaired iron homeostasis.

The Impact of ZIP8 Disease-Associated Variants G38R, C113S, G204C, and S335T on Selenium and Cadmium Accumulations: The First Characterization.

The metal cation symporter ZIP8 (SLC39A8) is a transmembrane protein that imports the essential micronutrients iron, manganese, and zinc, as well as heavy toxic metal cadmium (Cd). It has been recently suggested that selenium (Se), another essential micronutrient that has long been known for its role in human health and cancer risk, may also be transported by the ZIP8 protein. Several mutations in the ZIP8 gene are associated with the aberrant ion homeostasis of cells and can lead to human diseases. However, the intricate relationships between ZIP8 mutations, cellular Se homeostasis, and human diseases (including cancers and illnesses associated with Cd exposure) have not been explored. To further verify if ZIP8 is involved in cellular Se transportation, we first knockout (KO) the endogenous expression of ZIP8 in the HeLa cells using the CRISPR/Cas9 system. The elimination of ZIP8 expression was examined by PCR, DNA sequencing, immunoblot, and immunofluorescence analyses. Inductively coupled plasma mass spectrometry indicated that reduced uptake of Se, along with other micronutrients and Cd, was observed in the ZIP8-KO cells. In contrast, when ZIP8 was overexpressed, increased Se uptake could be detected in the ZIP8-overexpressing cells. Additionally, we found that ZIP8 with disease-associated single-point mutations G38R, G204C, and S335T, but not C113S, showed reduced Se transport ability. We then evaluated the potential of Se on Cd cytotoxicity prevention and therapy of cancers. Results indicated that Se could suppress Cd-induced cytotoxicity via decreasing the intracellular Cd transported by ZIP8, and Se exhibited excellent anticancer activity against not all but only selected cancer cell lines, under restricted experimental conditions. Moreover, clinical-based bioinformatic analyses revealed that up-regulated ZIP8 gene expression was common across multiple cancer types, and selenoproteins that were significantly co-expressed with ZIP8 in these cancers had been identified. Taken together, this study concludes that ZIP8 is an important protein in modulating cellular Se levels and provides insights into the roles of ZIP8 and Se in disease prevention and therapy.

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes.

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 µM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.

The solute carriers ZIP8 and ZIP14 regulate manganese accumulation in brain microvascular endothelial cells and control brain manganese levels.

Manganese supports numerous neuronal functions but in excess is neurotoxic. Consequently, regulation of manganese flux at the blood-brain barrier (BBB) is critical to brain homeostasis. However, the molecular pathways supporting the transcellular trafficking of divalent manganese ions within the microvascular capillary endothelial cells (BMVECs) that constitute the BBB have not been examined. In this study, we have determined that ZIP8 and ZIP14 (Zrt- and Irt-like proteins 8 and 14) support Mn2+ uptake by BMVECs and that neither DMT1 nor an endocytosis-dependent pathway play any significant role in Mn2+ uptake. Specifically, siRNA-mediated knockdown of ZIP8 and ZIP14 coincided with a decrease in manganese uptake, and kinetic analyses revealed that manganese uptake depends on pH and bicarbonate and is up-regulated by lipopolysaccharide, all biochemical markers of ZIP8 or ZIP14 activity. Mn2+ uptake also was associated with cell-surface membrane presentation of ZIP8 and ZIP14, as indicated by membrane protein biotinylation. Importantly, surface ZIP8 and ZIP14 biotinylation and Mn2+-uptake experiments together revealed that these transporters support manganese uptake at both the apical, blood and basal, brain sides of BMVECs. This indicated that in the BMVECs of the BBB, these two transporters support a bidirectional Mn2+ flux. We conclude that BMVECs play a critical role in controlling manganese homeostasis in the brain.

SLC39A8 gene encoding a metal ion transporter: discovery and bench to bedside.

SLC39A8 is an evolutionarily highly conserved gene that encodes the ZIP8 metal cation transporter in all vertebrates. SLC39A8 is ubiquitously expressed, including pluripotent embryonic stem cells; SLC39A8 expression occurs in every cell type examined. Uptake of ZIP8-mediated Mn2+, Zn2+, Fe2+, Se4+, and Co2+ represents endogenous functions-moving these cations into the cell. By way of mouse genetic differences, the phenotype of "subcutaneous cadmium-induced testicular necrosis" was assigned to the Cdm locus in the 1970s. This led to identification of the mouse Slc39a8 gene, its most closely related Slc39a14 gene, and creation of Slc39a8-overexpressing, Slc39a8(neo/neo) knockdown, and cell type-specific conditional knockout mouse lines; the Slc39a8(-/-) global knockout mouse is early-embryolethal. Slc39a8(neo/neo) hypomorphs die between gestational day 16.5 and postnatal day 1-exhibiting severe anemia, dysregulated hematopoiesis, hypoplastic spleen, dysorganogenesis, stunted growth, and hypomorphic limbs. Not surprisingly, genome-wide association studies subsequently revealed human SLC39A8-deficiency variants exhibiting striking pleiotropy-defects correlated with clinical disorders in virtually every organ, tissue, and cell-type: numerous developmental and congenital disorders, the immune system, cardiovascular system, kidney, lung, liver, coagulation system, central nervous system, musculoskeletal system, eye, and gastrointestinal tract. Traits with which SLC39A8-deficiency variants are currently associated include Mn2+-deficient hypoglycosylation; numerous birth defects; Leigh syndrome-like mitochondrial redox deficiency; decreased serum high-density lipoprotein-cholesterol levels; increased body mass index; greater risk of coronary artery disease, hypotension, cardiovascular death, allergy, ischemic stroke, schizophrenia, Parkinson disease, inflammatory bowel disease, Crohn disease, myopia, and adolescent idiopathic scoliosis; systemic lupus erythematosus with primary Sjögren syndrome; decreased height; and inadvertent participation in the inflammatory progression of osteoarthritis.

The Functions of ZIP8, ZIP14, and ZnT10 in the Regulation of Systemic Manganese Homeostasis.

As an essential nutrient, manganese is required for the regulation of numerous cellular processes, including cell growth, neuronal health, immune cell function, and antioxidant defense. However, excess manganese in the body is toxic and produces symptoms of neurological and behavioral defects, clinically known as manganism. Therefore, manganese balance needs to be tightly controlled. In the past eight years, mutations of genes encoding metal transporters ZIP8 (SLC39A8), ZIP14 (SLC39A14), and ZnT10 (SLC30A10) have been identified to cause dysregulated manganese homeostasis in humans, highlighting the critical roles of these genes in manganese metabolism. This review focuses on the most recent advances in the understanding of physiological functions of these three identified manganese transporters and summarizes the molecular mechanisms underlying how the loss of functions in these genes leads to impaired manganese homeostasis and human diseases.

Manganese Accumulation in the Brain via Various Transporters and Its Neurotoxicity Mechanisms.

Manganese (Mn) is an essential trace element, serving as a cofactor for several key enzymes, such as glutamine synthetase, arginase, pyruvate decarboxylase, and mitochondrial superoxide dismutase. However, its chronic overexposure can result in a neurological disorder referred to as manganism, presenting symptoms similar to those inherent to Parkinson's disease. The pathological symptoms of Mn-induced toxicity are well-known, but the underlying mechanisms of Mn transport to the brain and cellular toxicity leading to Mn's neurotoxicity are not completely understood. Mn's levels in the brain are regulated by multiple transporters responsible for its uptake and efflux, and thus, dysregulation of these transporters may result in Mn accumulation in the brain, causing neurotoxicity. Its distribution and subcellular localization in the brain and associated subcellular toxicity mechanisms have also been extensively studied. This review highlights the presently known Mn transporters and their roles in Mn-induced neurotoxicity, as well as subsequent molecular and cellular dysregulation upon its intracellular uptakes, such as oxidative stress, neuroinflammation, disruption of neurotransmission, α-synuclein aggregation, and amyloidogenesis.

Induction of the metal transporter ZIP8 by interferon gamma in intestinal epithelial cells: Potential role of metal dyshomeostasis in Crohn's disease.

Transition metals are required for intestinal homeostasis and provide essential nutrients for the resident microbiota. Abnormalities in metal homeostasis are common in Crohn's disease (CD), but remain poorly defined and causes appear multifactorial. There has been renewed interest in understanding these mechanisms with the discovery of an association between a coding variant in SLC39A8 (rs13107325; ZIP8 A391T) and increased CD risk. SLC39A8 encodes the protein ZIP8, a metal transporter that is induced under inflammatory stimuli; however, studies of its gut-specific functions are lacking. Here, we show that SLC39A8 mRNA is differentially expressed in active CD with a high positive correlation with markers of disease severity, including CXCL8, TNFα, IFNγ, and calprotectin. SLC39A8 expression exhibits a negative correlation with SLC39A4 and SLC39A5, two key zinc importers in absorptive enterocytes, and a lack of correlation with two manganese transporters, SLC39A14 and SLC11A2. Immunohistochemistry demonstrates ZIP8 expression in intestinal epithelial cells and immune cells of the lamina propria. Patients with CD exhibit variable patterns of ZIP8 subcellular localization within IECs. In ileal enteroids, SLC39A8 was induced by IFNγ and IFNγ + TNFα, but not by TNFα alone, independent of NF-κB activation. IFNγ also down-regulated SLC39A5. To explore the functional implications of disease-associated genetic variation, in over-expression experiments in HEK293A cells, ZIP8 A391T was associated with increased TNFα-induced NF-κB activation, consistent with a loss of negative regulation. Taken together, these results suggest a potential role for ZIP8 in intestinal inflammation, induced by IFNγ in the intestinal epithelial compartment, and that perturbations in negative regulation of NF-κB by ZIP8 A391T may contribute to CD pathogenesis.

Reduced hepatic metallothionein expression in first trimester fetuses in response to intrauterine smoking exposure: a consequence of low maternal zinc levels?

STUDY QUESTION: Does maternal smoking in early pregnancy affect metallothionein 1 and 2 (MT1 and MT2) mRNA and protein expression in first trimester placenta or embryonic/fetal liver? SUMMARY ANSWER: In the first trimester, MT protein expression is seen only in liver, where smoking is associated with a significantly reduced expression. WHAT IS KNOWN ALREADY: Zinc homeostasis is altered by smoking. Smoking induces MT in the blood of smokers properly as a result of the cadmium binding capacities of MT. In term placenta MT is present and smoking induces gene and protein expression (MT2 in particular), but the MT presence and response to smoking have never been examined in first trimester placenta or embryonic/fetal tissues. STUDY DESIGN, SIZE, DURATION: Cross sectional study where the presence of MT mRNA and protein was examined at the time of the abortion. The material was collected with informed consent after surgical intervention and frozen immediately. For protein expression analysis, liver tissue originating from smoking exposed n = 10 and unexposed n = 12 pregnancies was used. For mRNA expression analyses, placental tissue originating from smokers n = 19 and non-smokers n = 23 and fetal liver tissue from smoking exposed n = 16 and smoking unexposed pregnancies n = 13, respectively, were used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tissues were obtained from women who voluntarily and legally chose to terminate their pregnancy between gestational week 6 and 12. Western blot was used to determine the protein expression of MT, and real-time PCR was used to quantify the mRNA expression of MT2A and eight MT1 genes alongside the expression of key placental zinc transporters: zinc transporter protein-1 (ZNT1), Zrt-, Irt-related protein-8 and -14 (ZIP8 and ZIP14). MAIN RESULTS AND THE ROLE OF CHANCE: A significant reduction in the protein expression of MT1/2 in liver tissue (P = 0.023) was found by western blot using antibodies detecting both MT forms. Overall, a similar tendency was observed on the mRNA level although not statistically significant. Protein expression was not present in placenta, but the mRNA regulation suggested a down regulation of MT as well. A suggested mechanism based on the known role of MT in zinc homeostasis could be that the findings reflect reduced levels of easily accessible zinc in the blood of pregnant smokers and hence a reduced MT response in smoking exposed fetal/embryonic tissues. LIMITATIONS AND REASONS FOR CAUTION: Smoking was based on self-reports; however, our previous studies have shown high consistency regarding cotinine residues and smoking status. Passive smoking could interfere but was found mainly among smokers. The number of fetuses was limited, and other factors such as medication and alcohol might affect the findings. Information on alcohol was not consistently obtained, and we cannot exclude that it was more readily obtained from non-users. In the study, alcohol consumption was reported by a limited number (less than 1 out of 5) of women but with more smokers consuming alcohol. However, the alcohol consumption reported was typically limited to one or few times low doses. The interaction between alcohol and smoking is discussed in the paper. Notably we would have liked to measure zinc status to test our hypothesis, but maternal blood samples were not available. WIDER IMPLICATIONS OF THE FINDINGS: Zinc deficiency-in particular severe zinc deficiency-can affect pregnancy outcome and growth. Our findings indicate that zinc homeostasis is also affected in early pregnancy of smokers, and we know from pilot studies that even among women who want to keep their babies, the zinc status is low. Our findings support that zinc supplements should be considered in particular to women who smoke. STUDY FUNDING/COMPETING INTEREST(S): We thank the Department of Biomedicine for providing laboratory facilities and laboratory technicians and the Lundbeck Foundation and Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis Legat for financial support. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Role of ZIP8 in regulation of cisplatin sensitivity through Bcl-2.

ZIP8 is a membrane transporter that facilitates the uptake of divalent metals (e.g., Zn, Mn, Fe, Cd) and the mineral selenite in anionic form. ZIP8 functionality has been recently reported to regulate cell proliferation, migration and cytoskeleton arrangement, exhibiting an essential role for normal physiology. In this study, we report a ZIP8 role in chemotherapy response. We show ZIP8 regulates cell sensitivity to the anti-cancer drug cisplatin. Overexpression of ZIP8 in mouse embryonic fibroblast (MEF) cells induces cisplatin sensitivity, while knockout of ZIP8 in leukemia HAP1 cells leads to cisplatin resistance. In ZIP8 altered cells and transgenic mice, we show cisplatin is not a direct ZIP8 substrate. Further studies demonstrate that ZIP8 regulates anti-apoptotic protein Bcl-2. ZIP8 overexpression decreases Bcl-2 levels in cultured cells, mice lung and liver tissue while loss of ZIP8 elevates Bcl-2 expression in HAP1 cells and liver tissue. We also observe that ZIP8 overexpression modulates cisplatin-induced cell apoptosis, manifested by the increased protein level of cleaved Caspase-3. Since Bcl-2 elevation was previously discovered to induce cisplatin drug resistance, our results suggest ZIP8 may modulate cisplatin drug responses as well as apoptosis through Bcl-2. We therefore conclude ZIP8 is a new molecule to be involved in cisplatin drug responses and is predicted as a genetic factor to be considered in cisplatin therapy.

Iron uptake by ZIP8 and ZIP14 in human proximal tubular epithelial cells.

In patients with iron overload disorders, increasing number of reports of renal dysfunction and renal iron deposition support an association between increased iron exposure and renal injury. In systemic iron overload, elevated circulating levels of transferrin-bound (TBI) and non-transferrin-bound iron (NTBI) are filtered to the renal proximal tubules, where they may cause injury. However, the mechanisms of tubular iron handling remain elusive. To unravel molecular renal proximal tubular NTBI and TBI handling, human conditionally immortalized proximal tubular epithelial cells (ciPTECs) were incubated with 55Fe as NTBI and fluorescently labeled holo-transferrin as TBI. Ferrous iron importers ZIP8 and ZIP14 were localized in the ciPTEC plasma membrane. Whereas silencing of either ZIP8 or ZIP14 alone did not affect 55Fe uptake, combined silencing significantly reduced 55Fe uptake compared to control (p < 0.05). Furthermore, transferrin receptor 1 (TfR1) and ZIP14, but not ZIP8, colocalized with early endosome antigen 1 (EEA1). TfR1 and ZIP14 also colocalized with uptake of fluorescently labeled transferrin. Furthermore, ZIP14 silencing decreased 55Fe uptake after 55Fe-Transferrin exposure (p < 0.05), suggesting ZIP14 could be involved in early endosomal transport of TBI-derived iron into the cytosol. Our data suggest that human proximal tubular epithelial cells take up TBI and NTBI, where ZIP8 and ZIP14 are both involved in NTBI uptake, but ZIP14, not ZIP8, mediates TBI-derived iron uptake. This knowledge provides more insights in the mechanisms of renal iron handling and suggests that ZIP8 and ZIP14 could be potential targets for limiting renal iron reabsorption and enhancing urinary iron excretion in systemic iron overload disorders.