Shintaro Hojyo

Toll-like receptor–mediated regulation of zinc homeostasis influences dendritic cell function

Zinc is a trace element that is essential for the function of many enzymes and transcription factors. Zinc deficiency results in defects in innate and acquired immune responses. However, little is known about the mechanism(s) by which zinc affects immune cell function. Here we show that stimulation with the Toll-like receptor 4 agonist lipopolysaccharide (LPS) altered the expression of zinc transporters in dendritic cells and thereby decreased intracellular free zinc. A zinc chelator mimicked the effects of LPS, whereas zinc supplementation or overexpression of the gene encoding Zip6, a zinc transporter whose expression was reduced by LPS, inhibited LPS-induced upregulation of major histocompatibility complex class II and costimulatory molecules. These results establish a link between Toll-like receptor signaling and zinc homeostasis.

The Zinc Transporter SLC39A13/ZIP13 Is Required for Connective Tissue Development; Its Involvement in BMP/TGF-β Signaling Pathways

Background Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. Methodology/Principal Findings Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-β signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. Conclusions/Significance Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-β signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-β signaling and connective tissue dysfunction.

The diabetes-susceptible gene SLC30A8/ZnT8 regulates hepatic insulin clearance.

Recent genome-wide association studies demonstrated that common variants of solute carrier family 30 member 8 gene (SLC30A8) increase susceptibility to type 2 diabetes. SLC30A8 encodes zinc transporter-8 (ZnT8), which delivers zinc ion from the cytoplasm into insulin granules. Although it is well known that insulin granules contain high amounts of zinc, the physiological role of secreted zinc remains elusive. In this study, we generated mice with β cell-specific Slc30a8 deficiency (ZnT8KO mice) and demonstrated an unexpected functional linkage between Slc30a8 deletion and hepatic insulin clearance. The ZnT8KO mice had low peripheral blood insulin levels, despite insulin hypersecretion from pancreatic β cells. We also demonstrated that a substantial amount of the hypersecreted insulin was degraded during its first passage through the liver. Consistent with these findings, ZnT8KO mice and human individuals carrying rs13266634, a major risk allele of SLC30A8, exhibited increased insulin clearance, as assessed by c-peptide/insulin ratio. Furthermore, we demonstrated that zinc secreted in concert with insulin suppressed hepatic insulin clearance by inhibiting clathrin-dependent insulin endocytosis. Our results indicate that SLC30A8 regulates hepatic insulin clearance and that genetic dysregulation of this system may play a role in the pathogenesis of type 2 diabetes.

The Zinc Transporter SLC39A14/ZIP14 Controls G-Protein Coupled Receptor-Mediated Signaling Required for Systemic Growth

Aberrant zinc (Zn) homeostasis is associated with abnormal control of mammalian growth, although the molecular mechanisms of Zns roles in regulating systemic growth remain to be clarified. Here we report that the cell membrane-localized Zn transporter SLC39A14 controls G-protein coupled receptor (GPCR)-mediated signaling. Mice lacking Slc39a14 (Slc39a14-KO mice) exhibit growth retardation and impaired gluconeogenesis, which are attributable to disrupted GPCR signaling in the growth plate, pituitary gland, and liver. The decreased signaling is a consequence of the reduced basal level of cyclic adenosine monophosphate (cAMP) caused by increased phosphodiesterase (PDE) activity in Slc39a14-KO cells. We conclude that SLC39A14 facilitates GPCR-mediated cAMP-CREB signaling by suppressing the basal PDE activity, and that this is one mechanism for Zns involvement in systemic growth processes. Our data highlight SLC39A14 as an important novel player in GPCR-mediated signaling. In addition, the Slc39a14-KO mice may be useful for studying the GPCR-associated regulation of mammalian systemic growth.

Biochemical characterization of human ZIP13 protein: a homo-dimerized zinc transporter involved in the Spondylocheiro dysplastic Ehlers-Danlos syndrome

Background: ZIP13 protein is important for connective tissue development, which has not been characterized in detail. Results: ZIP13 is an eight-transmembrane protein with a unique hydrophilic region that forms a homo-dimer. Conclusion: ZIP13 is a homo-dimerized zinc transporter that possesses domains that are not found in other LZT families. Significance: The data and materials provide useful information and opportunity for further structural and functional analyses of ZIP13. The human SLC39A13 gene encodes ZIP13, a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) family. The ZIP13 protein is important for connective tissue development, and its loss of function is causative for the spondylocheiro dysplastic form of Ehlers-Danlos syndrome. However, this protein has not been characterized in detail. Here we report the first detailed biochemical characterization of the human ZIP13 protein using its ectopic expressed and the purified recombinant protein. Protease accessibility, microscopic, and computational analyses demonstrated that ZIP13 contains eight putative transmembrane domains and a unique hydrophilic region and that it resides with both its N and C termini facing the luminal side on the Golgi. Analyses including cross-linking, immunoprecipitation, Blue Native-PAGE, and size-exclusion chromatography experiments indicated that the ZIP13 protein may form a homo-dimer. We also demonstrated that ZIP13 mediates zinc influx, as assessed by monitoring the expression of the metallothionein gene and by detecting the intracellular zinc level with a zinc indicator, FluoZin-3. Our data indicate that ZIP13 is a homo-dimerized zinc transporter that possesses some domains that are not found in other LZT family members. This is the first biochemical characterization of the physiologically important protein ZIP13 and the demonstration of homo-dimerization for a mammalian ZIP zinc transporter family member. This biochemical characterization of the human ZIP13 protein provides important information for further investigations of its structural characteristics and function.

ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders

The liver, pancreas, and heart are particularly susceptible to iron-related disorders. These tissues take up plasma iron from transferrin or non-transferrin-bound iron, which appears during iron overload. Here, we assessed the effect of iron status on the levels of the transmembrane transporters, ZRT/IRT-like protein 14 and divalent metal-ion transporter-1, which have both been implicated in transferrin- and non-transferrin-bound iron uptake. Weanling male rats (n=6/group) were fed an iron-deficient, iron-adequate, or iron-overloaded diet for 3 weeks. ZRT/IRT-like protein 14, divalent metal-ion transporter-1 protein and mRNA levels in liver, pancreas, and heart were determined by using immunoblotting and quantitative reverse transcriptase polymerase chain reaction analysis. Confocal immunofluorescence microscopy was used to localize ZRT/IRT-like protein 14 in the liver and pancreas. ZRT/IRT-like protein 14 and divalent metal-ion transporter-1 protein levels were also determined in hypotransferrinemic mice with genetic iron overload. Hepatic ZRT/IRT-like protein 14 levels were found to be 100% higher in iron-loaded rats than in iron-adequate controls. By contrast, hepatic divalent metal-ion transporter-1 protein levels were 70% lower in iron-overloaded animals and nearly 3-fold higher in iron-deficient ones. In the pancreas, ZRT/IRT-like protein 14 levels were 50% higher in iron-overloaded rats, and in the heart, divalent metal-ion transporter-1 protein levels were 4-fold higher in iron-deficient animals. At the mRNA level, ZRT/IRT-like protein 14 expression did not vary with iron status, whereas divalent metal-ion transporter-1 expression was found to be elevated in iron-deficient livers. Immunofluorescence staining localized ZRT/IRT-like protein 14 to the basolateral membrane of hepatocytes and to acinar cells of the pancreas. Hepatic ZRT/IRT-like protein 14, but not divalent metal-ion transporter-1, protein levels were elevated in iron-loaded hypotransferrinemic mice. In conclusion, ZRT/IRT-like protein 14 protein levels are up-regulated in iron-loaded rat liver and pancreas and in hypotransferrinemic mouse liver. Divalent metal-ion transporter-1 protein levels are down-regulated in iron-loaded rat liver, and up-regulated in iron-deficient liver and heart. Our results provide insight into the potential contributions of these transporters to tissue iron uptake during iron deficiency and overload.

Zinc suppresses Th17 development via inhibition of STAT3 activation

Zinc (Zn) is an essential trace metal required by many enzymes and transcription factors for their activity or the maintenance of their structure. Zn has a variety of effects in the immune responses and inflammation, although it has not been well known how Zn affects these reactions on the molecular basis. We here showed that Zn suppresses T(h)17-mediated autoimmune diseases at lest in part by inhibiting the development of T(h)17 cells via attenuating STAT3 activation. In mice injected with type II collagen to induce arthritis, Zn treatment inhibited T(h)17 cell development. IL-6-mediated activation of STAT3 and in vitro T(h)17 cell development were all suppressed by Zn. Importantly, Zn binding changed the alpha-helical secondary structure of STAT3, disrupting the association of STAT3 with JAK2 kinase and with a phospho-peptide that included a STAT3-binding motif from the IL-6 signal transducer gp130. Thus, we conclude that Zn suppresses STAT3 activation, which is a critical step for T(h)17 development.

SLC39A14 Is Required for the Development of Hepatocellular Iron Overload in Murine Models of Hereditary Hemochromatosis

Nearly all forms of hereditary hemochromatosis are characterized by pathological iron accumulation in the liver, pancreas, and heart. These tissues preferentially load iron because they take up non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. Yet, how tissues take up NTBI is largely unknown. We report that ablation of Slc39a14, the gene coding for solute carrier SLC39A14 (also called ZIP14), in mice markedly reduced the uptake of plasma NTBI by the liver and pancreas. To test the role of SLC39A14 in tissue iron loading, we crossed Slc39a14(-/-) mice with Hfe(-/-) and Hfe2(-/-) mice, animal models of type 1 and type 2 (juvenile) hemochromatosis, respectively. Slc39a14 deficiency in hemochromatotic mice greatly diminished iron loading of the liver and prevented iron deposition in hepatocytes and pancreatic acinar cells. The data suggest that inhibition of SLC39A14 may mitigate hepatic and pancreatic iron loading and associated pathologies in iron overload disorders.

Zinc transporter SLC39A10/ZIP10 controls humoral immunity by modulating B-cell receptor signal strength.

Significance The essential micronutrient zinc is known to modulate adaptive immune responses and dysregulated zinc homeostasis leads to immunodeficiency. However, the molecular mechanisms underlying this zinc-mediated modulation are unknown. We show that the zinc transporter ZIP10 plays an important role in B-cell receptor (BCR) signaling. Zip10-deficiency in mature B cells attenuated both T-cell–dependent and –independent immune responses. Zip10-deficient mature B cells proliferated poorly in response to BCR cross-linking, as a result of dysregulated BCR signaling. Our data establish that ZIP10 functions as a cellular regulator to modulate BCR signaling in humoral immune responses. The humoral immune response, also called the antibody-mediated immune response, is one of the main adaptive immune systems. The essential micronutrient zinc (Zn) is known to modulate adaptive immune responses, and dysregulated Zn homeostasis leads to immunodeficiency. However, the molecular mechanisms underlying this Zn-mediated modulation are largely unknown. Here, we show that the Zn transporter SLC39A10/ZIP10 plays an important role in B-cell antigen receptor (BCR) signal transduction. Zip10-deficiency in mature B cells attenuated both T-cell–dependent and –independent immune responses in vivo. The Zip10-deficient mature B cells proliferated poorly in response to BCR cross-linking, as a result of dysregulated BCR signaling. The perturbed signaling was found to be triggered by a reduction in CD45R phosphatase activity and consequent hyperactivation of LYN, an essential protein kinase in BCR signaling. Our data suggest that ZIP10 functions as a positive regulator of CD45R to modulate the BCR signal strength, thereby setting a threshold for BCR signaling in humoral immune responses.

A novel role of the L-type calcium channel α1D subunit as a gatekeeper for intracellular zinc signaling: zinc wave.

Recent studies have shown that zinc ion (Zn) can behave as an intracellular signaling molecule. We previously demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI) rapidly release intracellular Zn from the endoplasmic reticulum (ER), and we named this phenomenon the “Zn wave”. However, the molecules responsible for releasing Zn and the roles of the Zn wave were elusive. Here we identified the pore-forming α1 subunit of the Cav1.3 (α1D) L-type calcium channel (LTCC) as the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, α1D was mainly localized to the ER rather than the plasma membrane in mast cells, and the Zn wave was impaired by α1D knockdown. We further found that the LTCC-mediated Zn wave positively controlled cytokine gene induction by enhancing the DNA-binding activity of NF- κB. Consistent with this finding, LTCC antagonists inhibited the cytokine-mediated delayed-type allergic reaction in mice without affecting the immediate-type allergic reaction. These findings indicated that the LTCC α1D subunit located on the ER membrane has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling and the delayed-type allergic reaction.