Maintenance of cellular zinc homeostasis includes regulating the expression of cell membrane zinc transporters. Knowledge about the mechanisms underlying changes in mammalian zinc transporter mRNA abundance is poor. We demonstrated that when expressed in Chinese hamster ovary cells as N-terminal fusions to green fluorescent protein, two splice variants of ZnT5 adopt different subcellular locations (either in the Golgi apparatus or throughout the cell, including at the plasma membrane) indicating discrete roles in cellular zinc homeostasis. We demonstrated, using a ␤-galactosidase reporter gene, that both splice variants were expressed from a promoter region that was transcriptionally repressed by increased extracellular zinc (150 M compared with 3 M; ϳ40%) and by extracellular zinc depletion, using the chelator N,N,N,N-tetrakis(2-pyridylmethyl) ethylenediamine (ϳ20%). We mapped the zinc-responsive element to the region ؊154 to ؉50, relative to the predicted start of transcription, and showed that a consensus metal response element sequence (؊410 to ؊404) was not responsible for these effects. Changes in ZnT5 mRNA abundance in Caco-2 cells at different zinc concentrations were in parallel to the changes in promoter activity (ϳ40% reduction at 150 M zinc) but in the presence of actinomycin D, to prevent transcription, we observed a marked stabilization (1.7-2-fold accumulation over 24 h) of ZnT5 mRNA. We conclude that effects of zinc on ZnT5 transcription and mRNA stability act in opposition to balance mRNA abundance for cellular zinc homeostasis. To our knowledge, this is the first report that zinc affects the stability of a transcript with a direct role in cellular zinc homeostasis.

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 coexpressed 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.

Only a small number of genes are known direct targets of the zinc-responsive transcription factor MTF1; therefore, the aim of this study was to gain a more complete understanding of the MTF-1 regulated zinc-responsive component of the transcriptome. A targeted siRNA was used to deplete MTF1 expression in the human intestinal cell line Caco-2. We predicted that the response to zinc of direct MTF1 target genes would be abrogated by MTF1 knockdown. Surprisingly, a greater number of genes were regulated by zinc following MFT1 knockdown, and most genes that responded to zinc under both control and MTF1-depleted conditions had an augmented response in the latter condition. Exceptions were the zinc effluxer ZnT1 and a suite of metallothionein genes, suggesting that responses of other genes to zinc are usually buffered by increases in these proteins. We propose that MTF1 heads a hierarchy of zinc sensors, and through controlling the expression of a raft of metallothioneins and other key proteins involved in controlling intracellular zinc levels (e.g. ZnT1) alters zinc buffering capacity and total cellular zinc content. We tested and validated this model by overexpressing metallothionein and observing the predicted curtailment in response of the zinc-repressed SLC30A5 (ZnT5) promoter. The model provides the framework for an integrated understanding of cellular zinc homeostasis. Because MTs can bind metals other than zinc, this framework links with overall cellular metal homeostasis.

Natural antisense transcripts (NATs) are important regulators of gene expression. Recently, a link between antisense transcription and the formation of endo-siRNAs has emerged. We investigated the bi-directionally transcribed Na/phosphate cotransporter gene (Slc34a1) under the aspect of endo-siRNA processing. Mouse Slc34a1 produces an antisense transcript that represents an alternative splice product of the Pfn3 gene located downstream of Slc34a1. The antisense transcript is prominently found in testis and in kidney. Co-expression of in vitro synthesized sense/antisense transcripts in Xenopus oocytes indicated processing of the overlapping transcripts into endo-siRNAs in the nucleus. Truncation experiments revealed that an overlap of at least 29 base-pairs is required to induce processing. We detected endo-siRNAs in mouse tissues that co express Slc34a1 sense/antisense transcripts by northern blotting. The orientation of endo-siRNAs was tissue specific in mouse kidney and testis. In kidney where the Na/phosphate cotransporter fulfils its physiological function endo-siRNAs complementary to the NAT were detected, in testis both orientations were found. Considering the wide spread expression of NATs and the gene silencing potential of endo-siRNAs we hypothesized a genome-wide link between antisense transcription and monoallelic expression. Significant correlation between random imprinting and antisense transcription could indeed be established. Our findings suggest a novel, more general role for NATs in gene regulation.