SLC-30A9 is required for Zn2+ homeostasis, Zn2+ mobilization, and mitochondrial health.

The trace element zinc is essential for many aspects of physiology. The mitochondrion is a major Zn2+ store, and excessive mitochondrial Zn2+ is linked to neurodegeneration. How mitochondria maintain their Zn2+ homeostasis is unknown. Here, we find that the SLC-30A9 transporter localizes on mitochondria and is required for export of Zn2+ from mitochondria in both Caenorhabditis elegans and human cells. Loss of slc-30a9 leads to elevated Zn2+ levels in mitochondria, a severely swollen mitochondrial matrix in many tissues, compromised mitochondrial metabolic function, reductive stress, and induction of the mitochondrial stress response. SLC-30A9 is also essential for organismal fertility and sperm activation in C. elegans, during which Zn2+ exits from mitochondria and acts as an activation signal. In slc-30a9-deficient neurons, misshapen mitochondria show reduced distribution in axons and dendrites, providing a potential mechanism for the Birk-Landau-Perez cerebrorenal syndrome where an SLC30A9 mutation was found.

Protective Effect of Znt7 on High Glucose-Induced Epithelial-to-Mesenchymal Transition in Renal Tubular Epithelial Cells.

BACKGROUND/AIMS: Evidence from our and other groups has demonstrated that zinc transporter 7 in SLC30 family (ZnT7) inhibited epithelial-to-mesenchymal transition (EMT) and apoptosis in rat peritoneal mesothelial cells (RPMCs) under high glucose (HG) concentration. In the present study, we investigated the effect of ZnT7 on EMT of renal tubular epithelial cells (RTECs) in an in vitro model of diabetic nephropathy (DN). METHODS: A dual-fluorescent staining protocol was used for detection of ZnT7 in a normal rat kidney tubular epithelial cell line (NRK-52E cells). EMT was induced with HG (30 mM). NRK-52E cells were transfected with plasmids codifying for hZnT7-EGFP and interfering RNA for determination of the effect of ZnT7 over-expression and silencing, respectively. Expression of ZnT7, activation of the MAPK/ERK and TGF-ß/Smad pathways were analyzed with by means of Western blot. RESULTS: ZnT7 was localized in the perinuclear region and Golgi apparatus. In HG-induced EMT of NRK-52E cells, ZnT7 was up-regulated. Over-expression of ZnT7 led to inhibition of HG-induced EMT, while knock-down of ZnT7 increased EMT. Furthermore, knock-down of ZnT7 and increased HG-induced EMT was accompanied by activation of the MAPK/ERK and TGF-ß/Smad pathways. CONCLUSION: The present study provides evidence that ZnT7 has a protective effect over EMT of RTECs in DN and suggests that the inhibition of HG-induced EMT may be achieved through the MAPK/ERK and TGF-ß/Smad pathways. Thereby, ZnT7 could be a potential target for translation medicine and prevention program in DN.

The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons.

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.

Nramp1 and NrampB Contribute to Resistance against Francisella in Dictyostelium.

The Francisella genus comprises highly pathogenic bacteria that can cause fatal disease in their vertebrate and invertebrate hosts including humans. In general, Francisella growth depends on iron availability, hence, iron homeostasis must be tightly regulated during Francisella infection. We used the system of the professional phagocyte Dictyostelium and the fish pathogen F. noatunensis subsp. noatunensis (F.n.n.) to investigate the role of the host cell iron transporters Nramp (natural resistance associated macrophage proteins) during Francisella infection. Like its mammalian ortholog, Dictyostelium Nramp1 transports iron from the phagosome into the cytosol, whereas the paralog NrampB is located on the contractile vacuole and controls, together with Nramp1, the cellular iron homeostasis. In Dictyostelium, Nramp1 localized to the F.n.n.-phagosome but disappeared from the compartment dependent on the presence of IglC, an established Francisella virulence factor. In the absence of Nramp transporters the bacteria translocated more efficiently from the phagosome into the host cell cytosol, its replicative niche. Increased escape rates coincided with increased proteolytic activity in bead-containing phagosomes indicating a role of the Nramp transporters for phagosomal maturation. In the nramp mutants, a higher bacterial load was observed in the replicative phase compared to wild-type host cells. Upon bacterial access to the cytosol of wt cells, mRNA levels of bacterial iron uptake factors were transiently upregulated. Decreased iron levels in the nramp mutants were compensated by a prolonged upregulation of the iron scavenging system. These results show that Nramps contribute to host cell immunity against Francisella infection by influencing the translocation efficiency from the phagosome to the cytosol but not by restricting access to nutritional iron in the cytosol.

MS-1 magA: Revisiting Its Efficacy as a Reporter Gene for MRI.

Bacterial genes involved in the biomineralization of magnetic nanoparticles in magnetotactic bacteria have recently been proposed as reporters for magnetic resonance imaging (MRI). In such systems, the expression of the bacterial genes in mammalian cells purportedly leads to greater concentrations of intracellular iron or the biomineralization of iron oxides, thus leading to an enhancement in relaxation rate that is detectable via MRI. Here, we show that the constitutive expression of the magA gene from Magnetospirillum magnetotacticum is tolerated by human embryonic kidney (HEK) cells but induces a strong toxic effect in murine mesenchymal/stromal cells and kidney-derived stem cells, severely restricting its effective use as a reporter gene for stem cells. Although it has been suggested that magA is involved in iron transport, when expressed in HEK cells, it does not affect the transcription of endogenous genes related to iron homeostasis. Furthermore, the magA-induced enhancement in iron uptake in HEK cells is insignificant, suggesting this gene is a poor reporter even for cell types that can tolerate its expression. We suggest that the use of magA for stem cells should be approached with caution, and its efficacy as a reporter gene requires a careful assessment on a cell-by-cell basis.

The zinc transporter Zip14 influences c-Met phosphorylation and hepatocyte proliferation during liver regeneration in mice.

BACKGROUND & AIMS: Zinc homeostasis in cells is maintained through tight regulation of zinc influx, efflux, and distribution to intracellular organelles by zinc transporters. The Zrt-Irt-like protein (ZIP) transporters facilitate zinc influx to the cytosol. Expression of the ZIP family member Zip14 can be induced by inflammatory cytokines, which also initiate liver regeneration. Hepatocyte proliferation is required for liver regeneration. Zinc regulates cell proliferation, tissue growth, and many mitogenic signaling pathways; we investigated its role in hepatocytes. METHODS: Wild-type and Zip14(-/-) mice that underwent partial hepatectomy (70% of liver removed) were used as models of liver regeneration. We also analyzed AML12 hepatocytes that overexpressed Zip14. Proliferation was assessed with proliferating cell nuclear antigen, CD1, and Ki67 markers and along with assays of zinc content was related to protein tyrosine phosphatase 1B (PTP1B) and extracellular signal-regulated kinase 1/2 signaling. RESULTS: Zip14 was up-regulated and hepatic zinc content increased during liver regeneration. Increased hepatic zinc inhibited activity of the phosphatase PTP1B and increased phosphorylation of c-Met, which promoted hepatocyte proliferation. AML12 cells that overexpressed Zip14 increased in zinc content and proliferation; PTP1B was inhibited and phosphorylation of c-Met increased. The increases in hepatic levels of zinc and hepatocyte proliferation that occurred following partial hepatectomy were not observed in Zip14(-/-) mice. CONCLUSIONS: The transporter Zip14 mediates hepatic uptake of zinc during liver regeneration and for hepatocyte proliferation. These findings indicate that zinc transporter activity regulates liver tissue growth by sequestering zinc. Reagents that regulate ZIP14 activity might be developed as therapeutics to promote liver regeneration in patients with chronic liver disease.

Regulation of glucagon secretion by zinc: lessons from the ß cell-specific Znt8 knockout mouse model.

In type-2 diabetes, hyperglucagonaemia aggravates elevated blood glucose levels. Relative to our knowledge of the ß-cell and insulin secretion, there remains a limited understanding of glucagon secretion in α-cells. Regulation of glucagon may be dependent on a combination of factors, which include direct glucose sensing by the α-cell, innervations from the autonomic nervous system and potential 'paracrine' actions by hormones and factors that are released by adjacent endocrine cells within the islets. The list of potential 'paracrine' regulators within the islet includes insulin, somatostatin, γ-aminobutyric acid, glutamate and zinc. Zinc crystallises with insulin in ß-cells and is co-secreted with insulin. In the scientific literature, the effect of exogeneous zinc on glucagon secretion has been debated. Here, we confirm that an increase in exogeneous zinc does inhibit glucagon secretion. To determine if there are physiological effects of zinc on glucagon secretion we used a ß-cell-specific ZnT8 knockout (Znt8BKO) mouse model. Znt8BKO mice, despite showing lower granular zinc content in ß-cells, showed no changes in fasted plasma glucagon levels and glucose regulated glucagon secretion. These findings suggest that zinc secreted from ß-cell does not regulate glucagon secretion.

The Drosophila copper transporter Ctr1C functions in male fertility.

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis.

Regulation of copper transporter 2 expression by copper and cisplatin in human ovarian carcinoma cells.

Down-regulation of copper transporter 1 (CTR1) reduces uptake and sensitivity, whereas down-regulation of CTR2 enhances both. Cisplatin (DDP) triggers the rapid degradation of CTR1 and thus limits its own accumulation. We sought to determine the effect of DDP and copper on the expression of CTR2. Changes in CTR1 and CTR2 mRNA and protein levels in human ovarian carcinoma 2008 cells and ATOX1(+/+) and ATOX1(-/-) mouse embryo fibroblasts in response to exposure to DDP and copper were measured by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and deconvolution microscopy. DDP triggered rapid degradation of CTR1 in 2008 human ovarian cancer cells. However, it increased the expression of CTR2 mRNA and protein levels. Expression of CTR2 was heavily modulated by changes in intracellular copper concentration; copper depletion produced rapid disappearance of CTR2, whereas excess copper increased the level of CTR2 protein. This increase was associated with an increase in CTR2 mRNA and prolongation of the CTR2 half-life. Consistent with prior observations that short hairpin RNA interference-mediated knockdown of CTR2 enhanced DDP uptake and tumor cell kill, reduction of CTR2 by copper starvation also enhanced DDP uptake and cytotoxicity. Comparison of the ability of copper and DDP to modulate the expression of CTR1 in ATOX1(+/+) and ATOX1(-/-) indicated that ATOX1 participates in the regulation of CTR2 expression. Unlike CTR1, the expression of CTR2 is increased rather than decreased by DDP. Therefore, these two copper transporters have opposite effects on DDP sensitivity. CTR2 expression is regulated by copper availability via the copper-dependent regulator ATOX1.

ZnT5 variant B is a bidirectional zinc transporter and mediates zinc uptake in human intestinal Caco-2 cells.

Zinc is an essential micronutrient, so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the functional properties of zinc transporters. Mammalian zinc transporters are classified in two major families: the SLC30 (ZnT) family and the SLC39 family. The prevailing view is that SLC30 family transporters function to reduce cytosolic zinc concentration, either through efflux across the plasma membrane or through sequestration in intracellular compartments, and that SLC39 family transporters function in the opposite direction to increase cytosolic zinc concentration. We demonstrated that human ZnT5 variant B (ZnT5B (hZTL1)), an isoform expressed at the plasma membrane, operates in both the uptake and the efflux directions when expressed in Xenopus laevis oocytes. We measured increased activity of the zinc-responsive metallothionein 2a (MT2a) promoter when ZnT5b was co-expressed with an MT2a promoter-reporter plasmid construct in human intestinal Caco-2 cells, indicating increased total intracellular zinc concentration. Increased cytoplasmic zinc concentration mediated by ZnT5B, in the absence of effects on intracellular zinc sequestration by the Golgi apparatus or endoplasmic reticulum, was also confirmed by a dramatically enhanced signal from the zinc fluorophore Rhodzin-3 throughout the cytoplasm of Caco-2 cells overexpressing ZnT5B at the plasma membrane when compared with control cells. Our findings demonstrate clearly that, in addition to mediating zinc efflux, ZnT5B at the plasma membrane can function to increase cytoplasmic zinc concentration, thus indicating a need to reevaluate the current paradigm that SLC30 family zinc transporters operate exclusively to decrease cytosolic zinc concentration.

[Effect of zinc on mRNA expression of ZIP4 in Caco2 cells].

OBJECTIVE: To study the effect of zinc on mRNA expression of ZIP4 in human intestinal Caco2 cells and its regularity. METHODS: Low zinc cell model was established by TPEN, a kind of chelating agent which chelates specially to zinc. ZIP4 cDNA fragment was obtained by RT-PCR. Expression of ZIP4 on 10 micromol/L TPEN exposure after 0, 2, 4, 6, 8 and 10 hours in Caco2 cells and its expression on various concentration of TPEN exposure (0,2.5,5,7.5 and 10 micromol/L) was measured by RT-PCR. RESULTS: A proper single fragment is obtained with the sequence conformable to the design. A proper single ZIP4 cDNA fragment was obtained. The mRNA expression of ZIP4 increased in accordance with the duration of low zinc. The peak mRNA level appeared at about 6h. And the ZIP4 mRNA increased in accordance with the concentration of TPEN in Caco2 cells. CONCLUSION: Zinc can regulate the mRNA expression of ZIP4 in Caco2 cells. And ZIP4 may play a role in the absorption of zinc in human intestine.

A novel pathway determining multidrug sensitivity in Schizosaccharomyces pombe.

In this study, we show that a mutation isolated during a screen for determinants of chemosensitivity in S. pombe results in loss of function of a previously uncharacterized protein kinase now named Hal4. Hal4 shares sequence homology to Hal4 and Hal5 in S. cerevisiae, and previous evidence indicates that these kinases positively regulate the major potassium transporter Trk1,2 and thereby maintain the plasma membrane potential. Disruption of this ion homeostasis pathway results in a hyperpolarized membrane and a concomitant increased sensitivity to cations. We demonstrate that a mutation in hal4+ results in hyperpolarization of the plasma membrane. In addition to the original selection agent, the hal4-1 mutant is sensitive to a variety of chemotherapeutic agents and stress-inducing compounds. Furthermore, this wider chemosensitive phenotype is also displayed by corresponding mutants in S. cerevisiae, and in a trk1deltatrk2delta double deletion mutant in S. pombe. We propose that this pathway and its role in regulating the plasma membrane potential may act as a pleiotropic determinant of sensitivity to chemotherapeutic agents.

Renal expression of osmotically responsive cation channel TRPV4 is restricted to water-impermeant nephron segments.

TRPV4, a nonselective cation channel of the transient receptor potential (TRP) family, is gated by hypotonicity. Expression of TRPV4 mRNA has been detected in the circumventricular organs of the brain responsible for sensing systemic tonicity and in the kidney distal convoluted tubule (DCT), among other sites. No analysis of TRPV4 expression at the protein level has been undertaken and no systematic analysis of expression of this channel has been reported in the kidney. Via RNAse protection assay and immunoblotting, abundant expression of TRPV4 was detected in the cortex, medulla, and papilla. The expression pattern of TRPV4 was characterized in both rat and mouse kidney, which revealed similar patterns of immunoreactivity. TRPV4 expression was absent from the proximal tubule (PT) and descending thin limb (DTL), whereas the strongest expression was observed in the ascending thin limb (ATL). The thick ascending limb (TAL) was strongly positive as was the DCT and connecting tubule. Importantly, the water-permeant cells of the macula densa were unstained. Moderate TRPV4 expression was noted in all collecting duct portions and in papillary epithelium; intercalated cells (type A) exhibited a particularly strong signal. In all positive segments, TRPV4 expression was concentrated at the basolateral membrane. Therefore, TRPV4 is expressed in only those nephron segments that are constitutively (i.e., ATL, TAL, and DCT) or conditionally (i.e., collecting duct) water impermeant and where generation of a substantial transcellular osmotic gradient could be expected. TRPV4 expression is absent from nephron segments exhibiting constitutive water permeability and unregulated apical aquaporin expression (i.e., PT and DTL). These data, although circumstantial, are consistent with a role for TRPV4 in the response to anisotonicity in the mammalian kidney.

Molecular mechanism of copper transport in Wilson disease.

Wilson disease is an autosomal recessive disorder of copper metabolism. The Wilson disease protein is a putative copper-transporting P-type ATPase, ATP7B, whose malfunction results in the toxic accumulation of copper in the liver and brain, causing the hepatic and/or neurological symptoms accompanying this disease. The cytosolic N-terminal domain (approximately 70 kDa) of this ATPase comprises six heavy metal-associated domains, each of which contains the conserved metal-binding motif GMTCXXC. The N-terminal domain (Wilson disease copper-binding domain [WCBD]) has been expressed, purified, and characterized using various techniques. The WCBD binds six atoms of copper in the +1 oxidation state competitively, and with a greater affinity than all other metals. The copper atom is coordinated by two cysteines in a distorted linear geometry. Copper binds the WCBD in a cooperative manner and induces secondary and tertiary conformation changes. Zinc binding to the WCBD has also been characterized by circular dichroism spectroscopy and shown to produce conformational changes that are completely different from those induced by copper. The phosphorylation/nucleotide-binding domain of ATP7B has also been expressed and characterized and shown to be capable of binding ATP but lacking ATPase activity. A peptide corresponding to the sixth transmembrane domain of ATP7B has been constructed and shown to undergo secondary conformational changes upon binding a single atom of copper. Finally, a chimeric protein consisting of the WCBD and truncated ZntA, a zinc-transporting ATPase lacking the N-terminal domain, has been constructed and analyzed for metal ion selectivity. These results suggest that the core determines the metal ion specificity of P-type ATPases, and the N-terminal metal-binding domain may play a regulatory role.