Hepatic metal ion transporter ZIP8 regulates manganese homeostasis and manganese-dependent enzyme activity.
ABSTRACT: Genetic variants at the solute carrier family 39 member 8 (SLC39A8) gene locus are associated with the regulation of whole-blood manganese (Mn) and multiple physiological traits. SLC39A8 encodes ZIP8, a divalent metal ion transporter best known for zinc transport. Here, we hypothesized that ZIP8 regulates Mn homeostasis and Mn-dependent enzymes to influence metabolism. We generated Slc39a8-inducible global-knockout (ZIP8-iKO) and liver-specific-knockout (ZIP8-LSKO) mice and observed markedly decreased Mn levels in multiple organs and whole blood of both mouse models. By contrast, liver-specific overexpression of human ZIP8 (adeno-associated virus-ZIP8 [AAV-ZIP8]) resulted in increased tissue and whole blood Mn levels. ZIP8 expression was localized to the hepatocyte canalicular membrane, and bile Mn levels were increased in ZIP8-LSKO and decreased in AAV-ZIP8 mice. ZIP8-LSKO mice also displayed decreased liver and kidney activity of the Mn-dependent enzyme arginase. Both ZIP8-iKO and ZIP8-LSKO mice had defective protein N-glycosylation, and humans homozygous for the minor allele at the lead SLC39A8 variant showed hypogalactosylation, consistent with decreased activity of another Mn-dependent enzyme, β-1,4-galactosyltransferase. In summary, hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes. This work provides a mechanistic basis for the association of SLC39A8 with whole-blood Mn, potentially linking SLC39A8 variants with other physiological traits.
Project description:ZIP8 is a metal transporter with a role in manganese (Mn) homeostasis. A common genetic variant in ZIP8 (rs13107325; A391T) ranks in the top 10 of pleiotropic SNPs identified in GWAS; A391T has associations with an increased risk of schizophrenia, obesity, Crohn's disease, and reduced blood Mn. Here, we used CRISPR/Cas9-mediated knockin (KI) to generate a mouse model of ZIP8 A391T (Zip8 393T-KI mice). Recapitulating the SNP association with blood Mn, blood Mn was reduced in Zip8 393T-KI mice. There was restricted abnormal tissue Mn homeostasis, with decreases in liver and kidney Mn and a reciprocal increase in biliary Mn, providing in vivo evidence of hypomorphic Zip8 function. Upon challenge in a chemically induced colitis model, male Zip8 393T-KI mice exhibited enhanced disease susceptibility. ZIP8 391-Thr associated with reduced triantennary plasma N-glycan species in a population-based cohort to define a genotype-specific glycophenotype hypothesized to be linked to Mn-dependent glycosyltransferase activity. This glycophenotype was maintained in a cohort of patients with Crohn's disease. These data and the pleiotropic disease associations with ZIP8 391-Thr suggest underappreciated roles of Mn homeostasis in complex human disease.
Project description:SLC39A8 encodes ZIP8, a divalent metal ion transporter. Mutations in the SLC39A8 gene are associated with congenital disorder of glycosylation type II and Leigh syndrome. Notably, affected patients with both disorders exhibited severe manganese (Mn) deficiency. The cellular function of human SLC39A8 (hSLC39A8) and the mechanisms by which mutations in this protein lead to human diseases are unclear. Herein, we show that hSLC39A8 mediates 54Mn uptake by the cells, and its expression is regulated by Mn. While expression of wild-type hSLC39A8 increased 54Mn uptake activity, disease-associated mutations abrogated the ability of the transporter to mediate Mn uptake into the cells, thereby providing a causal link to severe Mn deficiency. All mutants failed to localize on the cell surface and were retained within the endoplasmic reticulum. Interestingly, expression of hSLC39A8 mutants of both CDG type II and Leigh syndrome reduced mitochondrial 54Mn levels and activity of Mn-dependent mitochondrial superoxide dismutase MnSOD, and in turn increased oxidative stress. The expression of wild-type hSLC39A8, but not the disease-associated mutants, promoted mitochondrial functions. Moreover, loss of function analyses further corroborate hSLC39A8's critical role in mediating Mn uptake and mitochondrial function. Our results provide a potential pathogenic mechanism of diseases that are associated with hSLC39A8 mutations.
Project description:Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn(2+)/(HCO(3)(-))(2) symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects-proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.
Project description:Recent studies have demonstrated elevated levels of iron (Fe) in brains of patients with Huntington's disease (HD). Striatal cells carrying mutated Huntingtin presented increased sensitivity to cadmium (Cd) toxicity, decreased sensitivity to manganese (Mn) toxicity and deficits in Mn uptake. The hypothesis arose that the observed alterations result from the altered expression and/or activity of proteins engaged in the transport of these metals, that is: transferrin (TF), transferrin receptor (TFR), divalent metal transporter 1 (DMT1) and ZIP8 protein. Here we examined the expression levels of genes encoding these proteins in blood of HD patients and control subjects. A decreasing tendency in the level of TF transcript and increasing tendency of SLC11A2 mRNA encoding DMT1 was observed in the blood of HD patients compared to the control subjects, but neither attained statistical significance. No changes were found in the levels of TFRC coding for TFR and SLC39A8 coding for ZIP8 between HD patients and controls. The results indicate that HD-associated changes in metal homeostasis occur are not related to mechanisms other than the expression level of the here analyzed metal transporters.
Project description:Background: Manganese (Mn) is an essential element but at excessive levels, it is neurotoxic. Even a moderate increase in Mn has been suggested to interfere with neurodevelopment in children. Genetics influencing Mn concentrations and toxicity is unclear. Objective: We assessed, in a cross-sectional study, whether common single-nucleotide polymorphisms in the Mn transporters SLC39A8 (influx) and SLC30A10 (efflux) are associated with neurodevelopment in children. Design: We genotyped SLC39A8 (rs13107325 C/T) and SLC30A10 (rs1776029 G/A and rs12064812 T/C) in Italian children (n = 686, ages 11-14). We then used linear regression models to analyze associations between genotype, blood Mn concentrations, and neurodevelopmental outcomes including intelligence, behavior, motor function, and sway. Inferred causal relationships were evaluated using instrumental variables (IV) analysis. Results: For SLC30A10 rs1776029, the minor allele (A) was associated with increased average blood Mn of 41% (p < 0.001), whereas minor alleles for rs12064812 (C) and rs13107325 (T) were associated with reduced blood Mn of 7% (p = 0.002) and 15% (p < 0.001), respectively. For children carrying genotypes associated with high blood Mn, we observed lower performance for certain IQ subtests, increased sway, and increased scores for behavioral problems. High Mn genotypes showed odds ratios of 2-4 (p ? 0.01) for high scores in tests assessing ADHD-related behavior. IV analyses suggested that several of the associations were mediated by blood Mn. Conclusions: Our results suggest that common polymorphisms in SLC39A8 and SLC30A10 influence neurodevelopmental outcomes in children via differences in Mn homeostasis.
Project description:Previously this laboratory has identified the mouse Slc39a8 gene encoding the ZIP8 transporter, important in cadmium uptake. ZIP8 functions endogenously as a electroneutral Zn(2+)/(HCO(3)(-))(2) symporter, moving both ions into the cell. The overall physiological importance of ZIP8 remains unclear. Herein we describe generation of a mouse line carrying the Slc39a8(neo) allele, containing the Frt-flanked neomycin-resistance (neo) mini-cassette in intron 3 and loxP sites in introns 3 and 6. Cre recombinase functions correctly in Escherichia coli and in adeno-Cre-infected mouse fetal fibroblasts, but does not function in the intact mouse for reasons not clear. Slc39a8(neo) is a hypomorphic allele, because Slc39a8(neo/neo) homozygotes exhibit dramatically decreased ZIP8 expression in embryo, fetus, and visceral yolk sac - in comparison to their littermate wild-type controls. This ZIP8 hypomorph will be instrumental in studying developmental and in utero physiological functions of the ZIP8 transporter.
Project description:It has been known for decades that cadmium (Cd) must enter the cell to cause damage, but there was no mechanism to explain genetic differences in response to Cd toxicity until 2005. Starting with the mouse Cdm locus associated with differences in Cd-induced testicular necrosis between inbred strains, a 24.6-centiMorgan region on chromosome 3 was reduced ultimately to 880 kb; in this segment is the Slc39a8 gene encoding the ZIP8 Zn(2+)/HCO(3)(-) symporter. In endothelial cells of the testis vasculature, Cd-sensitive mice exhibit high ZIP8 expression, Cd-resistant mice exhibit very low expression. A 168.7-kb bacterial artificial chromosome (BAC) from a 129S6 (Cd-sensitive) BAC library containing the Slc39a8 gene was inserted into the Cd-resistant C57BL/6J genome: Cd treatment produced testicular necrosis in BAC-transgenic BTZIP8-3 mice but not in non-transgenic littermates, thereby proving that the Slc39a8 gene is indeed the Cdm locus. Cd-induced renal failure also occurred in these BTZIP8-3 mice. Immunohistochemistry showed highly expressed ZIP8 protein in the renal proximal tubular epithelial apical surface, suggesting that ZIP8 participates in Cd-induced renal failure. Slc39a14, most closely evolutionarily related to Slc39a8, encodes differentially-spliced products ZIP14A and ZIP14B that display properties similar to ZIP8. ZIP8 in alveolar cells brings environmental Cd into the organism and ZIP14 in intestinal enterocytes carries Cd into the organism and into the hepatocyte. We believe these two transporters function endogenously as Zn(2+)/HCO(3)(-) symporters important in combating inflammation and carrying out other physiological functions; Cd is able to displace the endogenous cation, enter the cell, and produce tissue damage and disease.
Project description:Slc39a8 encodes ZIP8, a divalent cation/bicarbonate symporter expressed in pluripotent mouse embryonic stem cells, and therefore ubiquitous in adult tissues; ZIP8 influxes Zn2+, Mn2+ and Fe2+. Slc39a8(neo/neo) knockdown mice exhibit 10-15% of wild-type ZIP8 mRNA and protein levels, and show pleiotropic phenotype of stunted growth, neonatal lethality, multi-organ dysmorphogenesis, and dysregulated hematopoiesis manifested as severe anemia. Herein we performed RNA-seq analysis of gestational day (GD)13.5 yolk sac and placenta, and GD16.5 liver, kidney, lung, heart and cerebellum, comparing Slc39a8(neo/neo) with Slc39a8(+/+) wild-type. Meta-data analysis of differentially-expressed genes revealed 29 unique genes from all tissues - having enriched GO categories associated with hematopoiesis and hypoxia and KEGG categories of complement, response to infection, and coagulation cascade - consistent with dysregulated hematopoietic stem cell fate. Based on transcription factor (TF) profiles in the JASPAR database, and searching for TF-binding sites enriched by Pscan, we identified numerous genes encoding zinc-finger and other TFs associated with hematopoietic stem cell functions. We conclude that, in this mouse model, deficient ZIP8-mediated divalent cation transport affects zinc-finger (e.g. GATA proteins) and other TFs interacting with GATA proteins (e.g. TAL1), predominantly in yolk sac. These data strongly support the phenotype of dysmorphogenesis and anemia seen in Slc39a8(neo/neo) mice in utero.
Project description:The alveolar epithelia of the lungs require manganese (Mn) as an essential nutrient, but also provide an entry route for airborne Mn that can cause neurotoxicity. Transporters involved in Mn uptake by alveolar epithelial cells are unknown. Recently, two members of the Zrt- and Irt-like protein (ZIP) family of metal transporters, ZIP8 and ZIP14, have been identified as crucial Mn importers in vivo. ZIP8 is by far most abundantly expressed in the lungs, whereas ZIP14 expression in the lungs is low compared to other tissues. We hypothesized that Mn uptake by alveolar epithelial cells is primarily mediated by ZIP8. To test our hypothesis, we used A549 cells, a type II alveolar cell line. Mirroring the in vivo situation, A549 cells expressed higher levels of ZIP8 than cell models for the liver, intestines, and kidney. Quantification of ZIP8 and ZIP14 revealed a strong enrichment of ZIP8 over ZIP14 in A549 cells. Using siRNA technology, we identified ZIP8 and ZIP14 as the major transporters mediating Mn uptake by A549 cells. To our surprise, knockdown of either ZIP8 or ZIP14 impaired Mn accumulation to a similar extent, which we traced back to similar amounts of ZIP8 and ZIP14 at the plasma membrane. Our study highlights the importance of both ZIP8 and ZIP14 in Mn metabolism of alveolar epithelial cells.
Project description:Trace elements such as copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) function as enzyme cofactors and second messengers in cell signaling. Trace elements are emerging as key regulators of differentiation and development of mammalian tissues including blood, brain, and skeletal muscle. We previously reported an influx of Cu and dynamic expression of metal transporters during differentiation of skeletal muscle cells. Here, we demonstrate that during differentiation of skeletal myoblasts an increase of Mn, Fe and Zn also occurs. Interestingly the Mn increase is concomitant with increased Mn-dependent SOD2 levels. To better understand the Mn import pathway in skeletal muscle cells, we probed the functional relevance of the closely related proteins ZIP8 and ZIP14, which are implicated in Zn, Mn, and Fe transport. Partial depletion of ZIP8 severely impaired growth of myoblasts and led to cell death under differentiation conditions, indicating that ZIP8-mediated metal transport is essential in skeletal muscle cells. Moreover, knockdown of Zip8 impaired activity of the Mn-dependent SOD2. Growth defects were partially rescued only by Mn supplementation to the medium, suggesting additional functions for ZIP8 in the skeletal muscle lineage. Restoring wild type Zip8 into the knockdown cells rescued the proliferation and differentiation phenotypes. On the other hand, knockdown of Zip14, had only a mild effect on myotube size, consistent with a role for ZIP14 in muscle hypertrophy. Simultaneous knockdown of both Zip8 and Zip14 further impaired differentiation and led cell death. This is the first report on the functional relevance of two members of the ZIP family of metal transporters in the skeletal muscle lineage, and further supports the paradigm that trace metal transporters are important modulators of mammalian tissue development.