ZIP Transporters Research Articles

Deletion of metal transporter Zip14 (Slc39a14) produces skeletal muscle wasting, endotoxemia, Mef2c activation and induction of miR-675 and Hspb7

Skeletal muscle represents the largest pool of body zinc, however, little is known about muscle zinc homeostasis or muscle-specific zinc functions. Zip14 (Slc39a14) was the most highly expressed zinc transporter in skeletal muscle of mice in response to LPS-induced inflammation. We compared metabolic parameters of skeletal muscle from global Zip14 knockout (KO) and wild-type mice (WT). At basal steady state Zip14 KO mice exhibited a phenotype that included muscle wasting and metabolic endotoxemia. Microarray and qPCR analysis of gastrocnemius muscle RNA revealed that ablation of Zip14 produced increased muscle p-Mef2c, Hspb7 and miR-675-5p expression and increased p38 activation. ChIP assays showed enhanced binding of NF-kappa beta to the Mef2c promoter. In contrast, LPS-induced systemic inflammation enhanced Zip14-dependent zinc uptake by muscle, increased expression of Atrogin1 and MuRF1 and markedly reduced MyoD. These signatures of muscle atrophy and cachexia were not influenced by Zip14 ablation, however. LPS-induced miR-675-3p and -5p expression was Zip14-dependent. Collectively, these results with an integrative model are consistent with a Zip14 function in skeletal muscle at steady state that supports myogenesis through suppression of metabolic endotoxemia and that Zip14 ablation coincides with sustained activity of phosphorylated components of signaling pathways including p-Mef2c, which causes Hspb7-dependent muscle wasting.

Imbalance in zinc homeostasis enhances lung Tissue Loss following cigarette smoke exposure

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease. Cadmium is a leading toxic component of cigarette smoke. Cadmium and zinc are highly related metals. Whereas, zinc is an essential metal required for normal health, cadmium is highly toxic. Zrt- and Irt-like protein 8 (ZIP8) is an avid transporter of both zinc and cadmium into cells and is abundantly expressed in the lung of smokers compared to nonsmokers. Our objective was to determine whether disturbed zinc homeostasis through diet or the zinc transporter ZIP8 increase susceptibility to lung damage following prolonged cigarette smoke exposure. MethodsCigarette smoke exposure was evaluated in the lungs of mice subject to insufficient and sufficient zinc intakes, in transgenic ZIP8 overexpressing mice, and a novel myeloid-specific ZIP8 knockout strain. ResultsModerate depletion of zinc intakes in adult mice resulted in a significant increase in lung cadmium burden and permanent lung tissue loss following prolonged smoke exposure. Overexpression of ZIP8 resulted in increased lung cadmium burden and more extensive lung damage, whereas cigarette smoke exposure in ZIP8 knockout mice resulted in increased lung tissue loss without a change in lung cadmium content, but a decrease in zinc. ConclusionsOverall, findings were consistent with past human studies. Imbalance in Zn homeostasis increases susceptibility to permanent lung injury following prolonged cigarette smoke exposure. Based on animal studies, both increased and decreased ZIP8 expression enhanced irreversible tissue damage in response to prolonged tobacco smoke exposure. We believe these findings represent an important advancement in our understanding of how imbalance in zinc homeostasis and cadmium exposure via tobacco smoke may increase susceptibility to smoking-induced lung disease.

Influence of dietary iron exposure on trace metal homeostasis and expression of metal transporters during development in zebrafish☆

The present study investigated the effects of dietary iron (Fe) exposure on physiological performance and homeostatic regulation of trace metals during development (5–28 days post-fertilization; dpf) in zebrafish (Danio rerio). The results demonstrated that whole body Fe content was increased in 14 dpf larvae fed a high Fe diet. Cumulative mortality was also significantly elevated during exposure to the high Fe diet. Using droplet digital PCR, we observed that high Fe-exposed larvae exhibited an increase in mRNA levels of the Fe-storage protein ferritin, which appeared to be associated with the elevated level of whole body Fe content. Further, the results indicated that dietary Fe exposure induced transient changes in the mRNA expression levels of various metal transporters, including the iron transporter dmt1, and the zinc transporters zip8 and zip14. The expression of the epithelial Ca2+ channels (i.e., ecac) was also found to increase by high dietary Fe. Overall, our findings suggest that larval fish during the early nutritional transition period are sensitive to the effects of dietary Fe.

Intestine-specific deletion of metal transporter Zip14 (Slc39a14) causes brain manganese overload and locomotor defects of manganism.

Impaired manganese (Mn) homeostasis can result in excess Mn accumulation in specific brain regions and neuropathology. Maintaining Mn homeostasis and detoxification is dependent on effective Mn elimination. Specific metal transporters control Mn homeostasis. Human carriers of mutations in the metal transporter ZIP14 and whole body Zip14-knockout (WB-KO) mice display similar phenotypes, including spontaneous systemic and brain Mn overload and motor dysfunction. Initially, it was believed that Mn accumulation due to ZIP14 mutations was caused by impaired hepatobiliary Mn elimination. However, liver-specific Zip14-KO mice did not show systemic Mn accumulation or motor deficits. ZIP14 is highly expressed in the small intestine and is localized to the basolateral surface of enterocytes. Thus, we hypothesized that basolaterally localized ZIP14 in enterocytes provides another route for the elimination of Mn. Using wild-type and intestine-specific Zip14-KO (I-KO) mice, we have shown that ablation of intestinal Zip14 is sufficient to cause systemic and brain Mn accumulation. The lack of intestinal ZIP14-mediated Mn excretion was compensated for by the hepatobiliary system; however, it was not sufficient to maintain Mn homeostasis. When supplemented with extra dietary Mn, I-KO mice displayed some motor dysfunctions and brain Mn accumulation based on both MRI imaging and chemical analysis, thus demonstrating the importance of intestinal ZIP14 as a route of Mn excretion. A defect in intestinal Zip14 expresssion likely could contribute to the Parkinson-like Mn accumulation of manganism.NEW & NOTEWORTHY Mn-induced parkinsonism is recognized as rising in frequency because of both environmental factors and genetic vulnerability; yet currently, there is no cure. We provide evidence in an integrative animal model that basolaterally localized ZIP14 regulates Mn excretion and detoxification and that deletion of intestinal ZIP14 leads to systemic and brain Mn accumulation, providing robust evidence for the indispensable role of intestinal ZIP14 in Mn excretion.

Elucidating the H+ Coupled Zn2+ Transport Mechanism of ZIP4; Implications in Acrodermatitis Enteropathica.

Cellular Zn2+ homeostasis is tightly regulated and primarily mediated by designated Zn2+ transport proteins, namely zinc transporters (ZnTs; SLC30) that shuttle Zn2+ efflux, and ZRT-IRT-like proteins (ZIPs; SLC39) that mediate Zn2+ influx. While the functional determinants of ZnT-mediated Zn2+ efflux are elucidated, those of ZIP transporters are lesser understood. Previous work has suggested three distinct molecular mechanisms: (I) HCO3− or (II) H+ coupled Zn2+ transport, or (III) a pH regulated electrodiffusional mode of transport. Here, using live-cell fluorescent imaging of Zn2+ and H+, in cells expressing ZIP4, we set out to interrogate its function. Intracellular pH changes or the presence of HCO3− failed to induce Zn2+ influx. In contrast, extracellular acidification stimulated ZIP4 dependent Zn2+ uptake. Furthermore, Zn2+ uptake was coupled to enhanced H+ influx in cells expressing ZIP4, thus indicating that ZIP4 is not acting as a pH regulated channel but rather as an H+ powered Zn2+ co-transporter. We further illustrate how this functional mechanism is affected by genetic variants in SLC39A4 that in turn lead to Acrodermatitis enteropathica, a rare condition of Zn2+ deficiency.

Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cells

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother's milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

Rat mandibular condyle and fossa grew separately then unified as a single joint at 20 days old, which was the weaning age.

Magnetic resonance imaging (MRI) was used to observe growth of the mandibular condyle, mandibular fossa, and articular disc as a single unit. Changes in each component's relative position and size were observed using 7-tesla MRI. Mandibular condyle chondrocytes' growth was evaluated with immunohistochemistry, using the expression of zinc transporter ZIP13. Three-dimensional T1-weighted (T1w) MRI was used to obtain images of the TMJ of Sprague Dawley rats at 4-78 days old (P4-78) with a voxel resolution of 65 μm. Two-dimensional T1w MR images were acquired after a subcutaneous injection of the contrast reagent gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). The T1w MR images showed that the mandibular condyle was located posterior to the mandibular fossa until P20; however, it then moved to a location underneath the mandibular fossa. In the Gd-DTPA enhanced images, the articular disc was identified as a region with lower signal intensity from P20. The number of ZIP13-positive chondrocytes at P6 was larger than the number at P24. In conclusion, the mandibular condyle with cartilage and disc grows on the posterior side of the mandibular fossa until P20, which was the weaning age. Then, the condyle fit into the mandibular fossa and completed the functional unit.

Molecular characterization of ten zinc (Zn) transporter genes and their regulation to Zn metabolism in freshwater teleost yellow catfish Pelteobagrus fulvidraco

BackgroundZn is an essential trace element for vertebrates, and Zn uptake and transport is related with the ZIP family of Zn transporters. Meantime, Zn also influenced the expression of ZIP family members. MethodsWe cloned and characterized the full-length cDNA sequences of ten Zn transport-relevant genes (ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14) from yellow catfish Pelteobagrus fulvidraco, investigated their mRNA tissue expression. These ZIP mRNA expression was also assessed in the primary hepatocytes and intestinal epithelial cells of yellow catfish in response to three Zn levels (0, 30 μM and 60 μM, respectively). ResultsAll these genes shared the similar domains with the corresponding members in mammals. The mRNA expression of the ten ZIP genes was detected in nine-tested tissues, but variable among these tissues. Flow cytometry analysis and confocal microscopy observation indicated that intracellular free Zn2+ concentration in hepatocytes and intestinal epithelial cells increased with increasing Zn incubation concentration at both 24 h and 48 h. Zn incubation differentially influenced mRNA levels of ZIP transporters in the hepatocytes and intestinal epithelial cells, in a time- and cells-dependent manners. In the hepatocytes, at 24 h, compared to the control, Zn addition down-regulated mRNA levels of ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP11 and ZIP14; however, ZIP10 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, mRNA levels of ZIP1, ZIP6, ZIP7, ZIP9, ZIP10 and ZIP14 declined with increasing Zn incubation concentrations; ZIP3 mRNA levels were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group. In the intestinal epithelial cells, at 24 h, Zn addition down-regulated mRNA levels of ZIP1, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14; ZIP3 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, Zn addition up-regulated mRNA levels of ZIP6 and ZIP9, but down-regulated mRNA levels of ZIP8, ZIP10 and ZIP13. ZIP7, ZIP11 and ZIP14 mRNA abundances were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group. ConclusionFor the first time, our study characterized ten ZIP family members in yellow catfish, explored their mRNA tissue expression. Their regulation to Zn addition were also investigated in the hepatocytes and intestinal epithelial cells of yellow catfish. Our study revealed the mechanism of cells exposed to Zn addition and provided novel insights for the regulatory mechanism of Zn homeostasis.

Loss of the dermis zinc transporter ZIP13 promotes the mildness of fibrosarcoma by inhibiting autophagy

Fibrosarcoma is a skin tumor that is frequently observed in humans, dogs, and cats. Despite unsightly appearance, studies on fibrosarcoma have not significantly progressed, due to a relatively mild tumor severity and a lower incidence than that of other epithelial tumors. Here, we focused on the role of a recently-found dermis zinc transporter, ZIP13, in fibrosarcoma progression. We generated two transformed cell lines from wild-type and ZIP13-KO mice-derived dermal fibroblasts by stably expressing the Simian Virus (SV) 40-T antigen. The ZIP13−/− cell line exhibited an impairment in autophagy, followed by hypersensitivity to nutrient deficiency. The autophagy impairment in the ZIP13−/− cell line was due to the low expression of LC3 gene and protein, and was restored by the DNA demethylating agent, 5-aza-2’-deoxycytidine (5-aza) treatment. Moreover, the DNA methyltransferase activity was significantly increased in the ZIP13−/− cell line, indicating the disturbance of epigenetic regulations. Autophagy inhibitors effectively inhibited the growth of fibrosarcoma with relatively minor damages to normal cells in xenograft assay. Our data show that proper control over autophagy and zinc homeostasis could allow for the development of a new therapeutic strategy to treat fibrosarcoma.

The effects of soil phosphorus and zinc availability on plant responses to mycorrhizal fungi: a physiological and molecular assessment

The positive effects of arbuscular mycorrhizal fungi (AMF) have been demonstrated for plant biomass, and zinc (Zn) and phosphorus (P) uptake, under soil nutrient deficiency. Additionally, a number of Zn and P transporter genes are affected by mycorrhizal colonisation or implicated in the mycorrhizal pathway of uptake. However, a comprehensive study of plant physiology and gene expression simultaneously, remains to be undertaken. Medicago truncatula was grown at different soil P and Zn availabilities, with or without inoculation of Rhizophagus irregularis. Measures of biomass, shoot elemental concentrations, mycorrhizal colonisation, and expression of Zn transporter (ZIP) and phosphate transporter (PT) genes in the roots, were taken. Mycorrhizal plants had a greater tolerance of both P and Zn soil deficiency; there was also evidence of AMF protecting plants against excessive Zn accumulation at high soil Zn. The expression of all PT genes was interactive with both P availability and mycorrhizal colonisation. MtZIP5 expression was induced both by AMF and soil Zn deficiency, while MtZIP2 was down-regulated in mycorrhizal plants, and up-regulated with increasing soil Zn concentration. These findings provide the first comprehensive physiological and molecular picture of plant-mycorrhizal fungal symbiosis with regard to soil P and Zn availability. Mycorrhizal fungi conferred tolerance to soil Zn and P deficiency and this could be linked to the induction of the ZIP transporter gene MtZIP5, and the PT gene MtPT4.

ZIP14 is degraded in response to manganese exposure

Manganese (Mn) is an essential element necessary for proper development and brain function. Circulating Mn levels are regulated by hepatobiliary clearance to limit toxic levels and prevent tissue deposition. To characterize mechanisms involved in hepatocyte Mn uptake, polarized human HepaRG cells were used for this study. Western blot analysis and immunofluorescence microscopy showed the Mn transporter ZIP14 was expressed and localized to the basolateral surface of polarized HepaRG cells. HepaRG cells took up 54Mn in a time- and temperature-dependent manner but uptake was reduced after exposure to Mn. This loss in transport activity was associated with decreased ZIP14 protein levels in response to Mn exposure. Mn-induced degradation of ZIP14 was blocked by bafilomycin A1, which increased localization of the transporter in Lamp1-positive vesicles. Mn exposure also down-regulated the Golgi proteins TMEM165 and GPP130 while the ER stress marker BiP was induced. These results indicate that Mn exposure decreases ZIP14 protein levels to limit subsequent uptake of Mn as a cytoprotective response. Thus, high levels of Mn may compromise first-pass-hepatic clearance mechanisms.

Activated zinc transporter ZIP7 as an indicator of anti-hormone resistance in breast cancer.

ZIP7, a member of the ZIP family of zinc importers, resides on the endoplasmic reticulum membrane and transports zinc from intracellular stores to the cytoplasm after activation by CK2 phosphorylation on two serine residues (S275 and S276). ZIP7 is known to be required for the growth of anti-hormone resistant breast cancer models, especially those with acquired tamoxifen resistance developed from MCF-7. Using our new pS275S276ZIP7 antibody which only recognises activated ZIP7 (pZIP7), we have demonstrated that the hyperactivation of ZIP7 is prevalent in tamoxifen-resistant breast cancer cells. This evidence suggests that pZIP7 might have potential as a biomarker of acquired resistance to such anti-hormones in breast cancer, a current unmet clinical need. In this regard, we have also developed a new immunohistochemical assay for pZIP7 which allowed pZIP7 to be tested on a small clinical series of breast cancer tissues confirming its prevalence in such tumours and relationship to a variety of clinicopathological parameters and biomarkers previously associated with endocrine resistant phenotypes, notably increased activated MAPK signalling, expression of ErbB2, CD71 and the proto-oncogene c-Fos, as well as with increased tumour grade.

Regulation of the Metal Transporters ZIP14 and ZnT10 by Manganese Intake in Mice.

The metal transporters ZIP14 and ZnT10 play key physiological roles in maintaining manganese (Mn) homeostasis. However, in vivo regulation of these two transporters by Mn is not understood. Here, we examined how dietary Mn intake regulates ZIP14 and ZnT10 by feeding mice a low-Mn diet, a control diet, or a high-Mn diet for 6 weeks. Inductively coupled plasma mass spectrometry was used to measure Mn and iron (Fe) levels. ZIP14 and ZnT10 protein levels were measured by western blot analysis. While mice on the high-Mn diet exhibited significantly higher levels of Mn in the blood, liver, and brain, the low-Mn diet group did not display matching reductions, indicating that high Mn intake is more effective in disrupting Mn homeostasis in mice. Additionally, Fe levels were only slightly altered, suggesting independent transport mechanisms for Mn and Fe. In the high-Mn diet group, ZIP14 and ZnT10 were both upregulated in the liver, as well as in the small intestine, indicating a coordinated role for these transporters in Mn excretion. Unexpectedly, this upregulation only occurred in male mice, with the exception of hepatic ZIP14, providing new insight into mechanisms behind widely observed sex differences in Mn homeostasis.

Inhibition of Zinc Transporter ZIP6/ZIP10 Heteromer Prevents the Zinc Influx Required to Trigger Mitosis

Zinc has been known to be essential for cell division for over 40 years, but the exact mechanism remains elusive. Cellular zinc import necessitates the help of the SLC39A family of ZIP transporters, two of which are known to be essential for gastrulation, which involves cell rounding and detachment. We have discovered a new molecular process that explains why zinc is required for cell division, involving these two zinc channels ZIP6 (previously LIV-1) and ZIP10 as a heteromer, providing cellular zinc entry to initiate the onset of mitosis. Crucially, blocking this ZIP6/ZIP10 heteromer-mediated zinc influx by specific antibodies completely prevents mitosis, even in the presence of mitosis causing agent nocodazole, confirming the requirement for this zinc influx as a mitosis trigger. This zinc influx additionally changes STAT3 phosphorylation to pS727STAT3, a poorly understood form of STAT3, stopping transcription factor activity and complexing with this heteromer throughout mitosis alongside pS38Stathmin, the form required for mitotic microtubule rearrangement. This discovery clarifies a role for ZIP6, ZIP10 and STAT3 in mitosis in addition to their known role in gastrulation, offering new opportunities for investigating the processes of cell development and cell division.

The Zinc Transporter Zip7 Is Downregulated in Skeletal Muscle of Insulin-Resistant Cells and in Mice Fed a High-Fat Diet.

Background: The zinc transporter Zip7 modulates zinc flux and controls cell signaling molecules associated with glucose metabolism in skeletal muscle. The present study evaluated the role of Zip7 in cell signaling pathways involved in insulin-resistant skeletal muscle and mice fed a high-fat diet. Methods: Insulin-resistant skeletal muscle cells were prepared by treatment with an inhibitor of the insulin receptor, HNMPA-(AM)3 or palmitate, and Zip7 was analyzed along with pAkt, pTyrosine and Glut4. Similarly, mice fed normal chow (NC) or a high-fat diet (HFD) were also analyzed for protein expression of Glut4 and Zip7. An overexpression system for Zip7 was utilized to determine the action of this zinc transporter on several genes implicated in insulin signaling and glucose control. Results: We identified that Zip7 is upregulated by glucose in normal skeletal muscle cells and downregulated in insulin-resistant skeletal muscle. We also observed (as expected) a decrease in pAkt and Glut4 in the insulin-resistant skeletal muscle cells. The overexpression of Zip7 in skeletal muscle cells led to the modulation of key genes involved in the insulin signaling axis and glucose metabolism including Akt3, Dok2, Fos, Hras, Kras, Nos2, Pck2, and Pparg. In an in vivo mouse model, we identified a reduction in Glut4 and Zip7 in the skeletal muscle of mice fed a HFD compared to NC controls. Conclusions: These data suggest that Zip7 plays a role in skeletal muscle insulin signaling and is downregulated in an insulin-resistant, and HFD state. Understanding the molecular mechanisms of Zip7 action will provide novel opportunities to target this transporter therapeutically for the treatment of insulin resistance and type 2 diabetes.

The zinc transporter Zip14 (SLC39a14) affects Beta-cell Function: Proteomics, Gene expression, and Insulin secretion studies in INS-1E cells

Insulin secretion from pancreatic beta-cells is dependent on zinc ions as essential components of insulin crystals, zinc transporters are thus involved in the insulin secretory process. Zip14 (SLC39a14) is a zinc importing protein that has an important role in glucose homeostasis. Zip14 knockout mice display hyperinsulinemia and impaired insulin secretion in high glucose conditions. Endocrine roles for Zip14 have been established in adipocytes and hepatocytes, but not yet confirmed in beta-cells. In this study, we investigated the role of Zip14 in the INS-1E beta-cell line. Zip14 mRNA was upregulated during high glucose stimulation and Zip14 silencing led to increased intracellular insulin content. Large-scale proteomics showed that Zip14 silencing down-regulated ribosomal mitochondrial proteins, many metal-binding proteins, and others involved in oxidative phosphorylation and insulin secretion. Furthermore, proliferation marker Mki67 was down-regulated in Zip14 siRNA-treated cells. In conclusion, Zip14 gene expression is glucose sensitive and silencing of Zip14 directly affects insulin processing in INS-1E beta-cells. A link between Zip14 and ribosomal mitochondrial proteins suggests altered mitochondrial RNA translation, which could disturb mitochondrial function and thereby insulin secretion. This highlights a role for Zip14 in beta-cell functioning and suggests Zip14 as a future pharmacological target in the treatment of beta-cell dysfunction.

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.

Novel role of zinc homeostasis in IL-23 regulation and host defense following bacterial infection

Abstract Zinc homeostasis is crucial for immune cell activation and intracellular zinc is tightly regulated by zinc transporters. Zinc deficiency is associated with increased prevalence of respiratory bacterial infections. Although the association between zinc and immunity is well established, the mechanisms by which zinc protects against bacteria remain to be elucidated. In human monocytes the zinc transporter Zip8 regulates inflammation in a zinc-dependent manner after LPS exposure. Given the critical role of APCs (macrophages and dendritic cells) in guiding the immune response against bacteria, we sought to determine the role of Zip8 in coordinating the immune response after bacterial exposure. In a novel myeloid-specific Zip8KO mouse model we observed that intranasal instillation of S. pneumoniae resulted in a significant increase of infiltrating neutrophils in the airways of Zip8KO mice when compared to WT counterparts. Analysis of BAL fluid revealed elevated levels of CXCL1 and IL-23 in the Zip8KO mice. To determine whether dendritic cells potentially play a role in the observed response, BMDCs were generated from Zip8KO and WT mice. LPS stimulation produced mature BMDCs with high CD11c and MHC-II expression in both groups. No significant changes were observed in the production of most cytokines examined (TNF-α, CXCL1, IL-6, IL-12), except for IL-23 which was consistently increased in the stimulated Zip8KO cells in comparison to WT control DCs. These results potentially reveal a vital axis that couples zinc homeostasis to IL-23 production in myeloid cells and ultimately host defense against invasive bacterial pathogens. To what extent this alters pathogen clearance and host recovery is the focus of future studies.

Unraveling the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2)

The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–translocation pore. Interestingly, the H63A substitution completely abrogated pH sensitivity, and substitutions of Glu-67 and Phe-269 altered the pH and voltage modulation of transport. In contrast, substitution of Glu-106, which might be part of a dimerization interface, altered pH but not voltage modulation. Substitution of Phe-269, located close to the substrate-binding site, also affected substrate selectivity. These findings were supported by an additional model of ZIP2 that was based on the structure of a prokaryotic homolog, Bordetella bronchiseptica ZrT/Irt-like protein (bbZIP), and in silico pKa calculations. We also found that residues Glu-179, His-175, His-202, and Glu-276 are directly involved in the coordination of the substrate metal ion. We noted that, unlike bbZIP, human ZIP2 is predicted to harbor a single divalent metal-binding site, with the charged side chain of Lys-203 replacing the second bound ion. Our results provide the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport, identify the substrate-binding site, and suggest a structure-based transport mechanism for the ZIP2 transporter.

Dietary Zinc Supplementation from Organic and Inorganic Sources Induce Differential Intestinal Gene Expression in Swine

BackgroundZinc (Zn) is an essential mineral supplemented to animal diets from organic (bound to organic compounds) and inorganic sources (inorganic salts). Diets supplemented with organic Zn result in better animal performance than inorganic sources especially under challenges like heat stress. The mechanisms by which sources of Zn differ in their physiological effects are unknown. A study in piglets showed that organic Zn sources increase mRNA expression of Zn transporters compared to inorganic Zn. A cell line study demonstrated that amino acid‐bound Zn can be absorbed by amino acid transporters, increasing Zn bioavailability in a model of acrodermatitis enteropathica. Here, we used in vitro and in vivo models to test the hypothesis that intestinal luminal exposure to organic and inorganic Zn sources differentially affects nutrient absorption and transport in the intestine.MethodsForty‐five pigs (body weight = 77.5 ± 2.5 kg) were allocated to pens (9 pigs/pen) and randomly assigned to five different diets: a corn‐soybean meal basal diet without added Zn (control diet), or control diet supplemented with 50 ppm (low) and 100 ppm (high) of ZnCl2 and amino acid‐bound Zn (LQ, Zinpro Corp.) as inorganic and organic sources respectively. After 32 days, ileum samples were collected and RNA was extracted for RNAseq analysis. To define if responses to Zn are due to luminal or basal signaling, mouse enteroids were microinjected with 50 ppm, 100 ppm, 150 ppm and 1000 ppm of either ZnCl2 or LQ‐Zn to model luminal exposure or Zn sources were added to the media to identify changes induced by circulating Zn. Enteroid gene expression of relevant genes was measured by qPCR.ResultsCompared to control diet, ZIP4 expression was increased by high and low LQ‐Zn and high ZnCl2, while low ZnCl2 induced ZIP10 expression. High ZnCl2 diet induced expression of SLC2A6 (GLUT6) compared with control. Comparison of inorganic to organic Zn sources showed 292 differentially expressed genes (DEG's) between low ZnCl2 (n=8) and low LQ‐Zn (n=9) and 370 DEG's between high ZnCl2 (n=8) and high LQ‐Zn (n=8). Of note, phospholipase A2 expression was repressed by high ZnCl2 compared to high LQ‐Zn. Aminopeptidase‐N, metallothionein‐1A (MT1), vitamin D 25‐ hydroxylase, interleukin 33 and epidermal growth factor receptor substrate 15‐like 1 genes showed higher expression induced by ZnCl2 compared to LQ‐Zn. Enteroids luminally exposed to ZnCl2 showed increased expression of metallothionein, Sglt1 and amino acid transporter Pept1. Both ZnCl2 and LQ‐Zn increased the expression of zinc transporter Zip7 in the enteroids. Zn in the enteroid media increased the expression of Glut2, ZnT1 and Mt1, and decreased the expression of Sglt1, Pept1 and Zip 4 compared with no additional Zn. However, no differences were found between ZnCl2 and LQ‐Zn treatments.ConclusionsOur results suggest that Zn supplementation modulates the intestinal expression of nutrients and Zn transporters. Findings in our enteroid experiment suggest that the changes are dependent on the source of Zn only when Zn is supplemented luminally. Overall, our findings provide information on the potential mechanisms by which Zn sources can affect animal performance and health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.