Angiotensin II Requires Zinc and Downregulation of the Zinc Transporters ZnT3 and ZnT10 to Induce Senescence of Vascular Smooth Muscle Cells

Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells

Peptide-Based, Two-Fluorophore, Ratiometric Probe for Quantifying Mobile Zinc in Biological Solutions

Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology. Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications. Using solid-phase peptide synthesis, we conjugated a zinc-sensitive Zinpyr-1 derivative and a zinc-insensitive 7-hydroxycoumarin derivative onto opposite ends of a rigid P₉K peptide scaffold to create HcZ9, a ratiometric fluorescent probe for mobile zinc. A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy. We investigated zinc accumulation in prostatic cells and zinc levels in human seminal fluid. When normal and tumorigenic cells are bathed in zinc-enriched media, cellular mobile zinc is buffered and changes slightly, but total zinc levels increase significantly. Quantification of mobile and total zinc levels in human seminal plasma revealed that the two are positively correlated with a Pearson’s coefficient of 0.73.National Institute of General Medical Sciences (U.S.) (GM065519

ttm-1 encodes CDF transporters that excrete zinc from intestinal cells of C. elegans and act in a parallel negative feedback circuit that promotes homeostasis

Zinc is an essential metal involved in a wide range of biological processes, and aberrant zinc metabolism is implicated in human diseases. The gastrointestinal tract of animals is a critical site of zinc metabolism that is responsible for dietary zinc uptake and distribution to the body. However, the role of the gastrointestinal tract in zinc excretion remains unclear. Zinc transporters are key regulators of zinc metabolism that mediate the movement of zinc ions across membranes. Here, we identified a comprehensive list of 14 predicted Cation Diffusion Facilitator (CDF) family zinc transporters in Caenorhabditis elegans and demonstrated that zinc is excreted from intestinal cells by one of these CDF proteins, TTM-1B. The ttm-1 locus encodes two transcripts, ttm-1a and ttm-1b, that use different transcription start sites. ttm-1b expression was induced by high levels of zinc specifically in intestinal cells, whereas ttm-1a was not induced by zinc. TTM-1B was localized to the apical plasma membrane of intestinal cells, and analyses of loss-of-function mutant animals indicated that TTM-1B promotes zinc excretion into the intestinal lumen. Zinc excretion mediated by TTM-1B contributes to zinc detoxification. These observations indicate that ttm-1 is a component of a negative feedback circuit, since high levels of cytoplasmic zinc increase ttm-1b transcript levels and TTM-1B protein functions to reduce the level of cytoplasmic zinc. We showed that TTM-1 isoforms function in tandem with CDF-2, which is also induced by high levels of cytoplasmic zinc and reduces cytoplasmic zinc levels by sequestering zinc in lysosome-related organelles. These findings define a parallel negative feedback circuit that promotes zinc homeostasis and advance the understanding of the physiological roles of the gastrointestinal tract in zinc metabolism in animals

Zn tolerance in the evergreen shrub, Aucuba japonica, naturally growing at a mine site: Cell wall immobilization, aucubin production, and Zn adsorption on fungal mycelia

journal articl

Functional analysis of AtZIP4, AtZIP6 and AtZIP9 metal transporters of Arabidopsis thaliana and Expression of Saccharomyces cerevisiae ZRC1 in different plant species.

Plants have developed a variety of adaptive strategies to take up sufficient quantities of essential macro- and micro-nutrients and avoid their excessive accumulation, which could be toxic. Metal transporters play an essential role in this homeostatic network by controlling the metal efflux across cellular membranes and compartments. The ZIP (ZRT IRT1-like Proteins: Zinc-regulated transporter Iron-regulated transporter 1-like protein) family of metal transporter is involved in this complex network. We have focused on three ZIP-family members: AtZIP4, AtZIP6 and AtZIP9, for which very little information is available. These genes have not been yet characterized although a possible role in micronutrients vascular system uploading and root uptake was hypothesized. ZIP6 is not part of the four main groups in the ZIP family and Milner et al., (2013) have highlighted its higher expression in roots than in shoots, whereas no expression modulation at varying Zn and Fe concentrations (Wintz et al., 2003). The transcript level of ZIP6 seems to be constitutively higher in the Zn hyperaccumulator Arabidopsis halleri ssp. halleri than in A. thaliana, independently of the Zn concentration (Becher et al., 2003). ZIP9 is clustered together with ZIP4, sharing c.a. 60% identity and 77% similarity at the amino acid sequence. Wintz et al. (2003) have shown that in A. thaliana both ZIP4 and ZIP9 are up-regulated in roots and shoots in Zn-deficient conditions. In A. halleri roots, ZIP9 is up-regulated upon Zn-deficiency and down-regulated in presence of high Zn concentrations. In A. thaliana shoots, ZIP9 level is detectable only upon excess Zn (Weber et al., 2004). The aim of this project is the functional analysis of AtZIP6 and AtZIP9, to understand their expression pattern and their subcellular localization. AtZIP6 and AtZIP9 knock-out mutant lines were investigated and will be used for testing metal tolerance and accumulation in order to identify which metal(s) are transported by these proteins. The high similarity between ZIP9 and ZIP4 prompted us to add AtZIP4 mutant our analysis to avoid a possible functional complementation. Furthermore, over-expressing A. thaliana lines have also been obtained fusing the 35S constitutive promoter to the AtZIP6 and AtZIP9 coding sequence in order to perform a phenotypic characterization and a metal accumulation analysis in comparison to single zip4, zip6, zip9, double zip6/zip9, zip4/zip9, zip4/zip6 and a triple zip4/zip6/zip9 knock-out mutants and wild-type lines Another part of the project concerns the effect of heterologous yeast genes in A. thaliana, tobacco and poplar plants, analyzing their ability in heavy metal accumulation. ZRC1 gene was selected since it induces Zn/Cd resistance in Saccharomyces cerevisiae. ScZRC1 is a vacuolar transporter that mediates the detoxification of Zn excess storing it into the vacuole (MacDiarmid et al., 2003). ScZRC1 belongs to the CDF transporter family and might have also a putative role in Cd detoxification

IL-4 Induces Metallothionein 3- and SLC30A4-Dependent Increase in Intracellular Zn2+ that Promotes Pathogen Persistence in Macrophages

SummaryAlternative activation of macrophages promotes wound healing but weakens antimicrobial defenses against intracellular pathogens. The mechanisms that suppress macrophage function to create a favorable environment for pathogen growth remain elusive. We show that interleukin (IL)-4 triggers a metallothionein 3 (MT3)- and Zn exporter SLC30A4-dependent increase in the labile Zn2+ stores in macrophages and that intracellular pathogens can exploit this increase in Zn to survive. IL-4 regulates this pathway by shuttling extracellular Zn into macrophages and by activating cathepsins that act on MT3 to release bound Zn. We show that IL-4 can modulate Zn homeostasis in both human monocytes and mice. In vivo, MT3 can repress macrophage function in an M2-polarizing environment to promote pathogen persistence. Thus, MT3 and SLC30A4 dictate the size of the labile Zn2+ pool and promote the survival of a prototypical intracellular pathogen in M2 macrophages

Imaging mobile zinc in biology

Trafficking and regulation of mobile zinc pools influence cellular functions and pathological conditions in multiple organs, including brain, pancreas, and prostate. The quest for a dynamic description of zinc distribution and mobilization in live cells fuels the development of increasingly sophisticated probes. Detection systems that respond to zinc binding with changes of their fluorescence emission properties have provided sensitive tools for mobile zinc imaging, and fluorescence microscopy experiments have afforded depictions of zinc distribution within live cells and tissues. Both small-molecule and protein-based fluorescent probes can address complex imaging challenges, such as analyte quantification, site-specific sensor localization, and real-time detection.National Institute of General Medical Sciences (U.S.) (grant GM065519