Jodi Dufner-Beattie

The Acrodermatitis Enteropathica Gene ZIP4 Encodes a Tissue-specific, Zinc-regulated Zinc Transporter in Mice

The human ZIP4 gene (SLC39A4) is a candidate for the genetic disorder of zinc metabolism acrodermatitis enteropathica. To understand its role in zinc homeostasis, we examined the function and expression of mouse ZIP4. This gene encodes a well conserved eight-transmembrane protein that can specifically increase the influx of zinc into transfected cells. Expression of this gene is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by zinc. Dietary zinc deficiency causes a marked increase in the accumulation of ZIP4 mRNA in these tissues, whereas injection of zinc or increasing zinc content of the diet rapidly reduces its abundance. Zinc can also regulate the accumulation of ZIP4 protein at the apical surface of enterocytes and visceral endoderm cells. These results provide compelling evidence that ZIP4 is a zinc transporter that plays an important role in zinc homeostasis, a process that is defective in acrodermatitis enteropathica in humans.

Structure, function, and regulation of a subfamily of mouse zinc transporter genes.

Zinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eight-transmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of zinc in transfected cultured cells, similar to the previously demonstrated functions of human ZIP1 and ZIP2 (Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560–5564; Gaither, L. A., and Eide, D. J. (2001) J. Biol. Chem. 276, 22258–22264). No ZIP3 orthologs have been previously studied. Furthermore, this first systematic comparative study of the in vivo expression and dietary zinc regulation of this subfamily of zip genes revealed that 1) zip1 mRNA is abundant in many mouse tissues, whereas zip2 and zip3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted in their accumulation; and 2) unlike mouse metallothionein I and zip4 mRNAs (Dufner-Beattie, J., Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D., and Andrews, G. K. (2003) J. Biol. Chem. 278, 33474–33481), the abundance of zip1, zip2, and zip3 mRNAs is not regulated by dietary zinc in the intestine and visceral endoderm, tissues involved in nutrient absorption. These studies suggest that all three of these ZIP proteins may play cell-specific roles in zinc homeostasis rather than primary roles in the acquisition of dietary zinc.

Novel zinc-responsive post-transcriptional mechanisms reciprocally regulate expression of the mouse Slc39a4 and Slc39a5 zinc transporters (Zip4 and Zip5).

Abstract Dietary zinc deficiency in mice is accompanied by enhanced expression of the zinc uptake transporter Slc39a4 (Zip4) and repressed expression of Slc39a5 (Zip5) in tissues which regulate zinc homeostasis (intestine, pancreas and visceral yolk sac). Herein, mechanisms controlling this differential expression were investigated. The induction of Zip4 mRNA during zinc deficiency, and its repression in response to zinc repletion were found to reflect changes in Zip4 mRNA stability and not changes in the relative rate of transcription of this gene. During zinc deficiency, ZIP4 protein levels are increased and this protein is localized on the apical membranes. Administration of an oral gavage of zinc caused ZIP4 internalization and degradation in enterocytes and visceral endoderm cells. Similarly, ZIP4 is induced by zinc deficiency in cultured mouse Hepa cells and is rapidly degraded in response to added zinc. Zip5 mRNA abundance does not change in response to zinc, but the translation of this mRNA was found to be zinc-responsive. During zinc deficiency, Zip5 mRNA remains associated with polysomes, while the protein is internalized and degraded in enterocytes, acinar cells and endoderm cells. After zinc-gavage, ZIP5 is rapidly resynthesized and targeted to the basolateral membranes of these cell types.

Generation and Characterization of Mice Lacking the Zinc Uptake Transporter ZIP3

ABSTRACT The mouse ZIP3 (SLC39A3) gene encodes an eight-transmembrane-domain protein that has been conserved in mammals and can function to transport zinc. To analyze the expression of ZIP3 in the early embryo and neonate and to determine its in vivo function, we generated ZIP3 null mice in which the ZIP3 open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP) reporter. EGFP fluorescence revealed that ZIP3 was expressed in the inner cell mass of the blastocyst and later during embryonic development in many tissues. Elevated expression was apparent in the embryonic brain and neurotube and neonatal gonads. Homozygous knockout mice were viable and fertile and under normal growth conditions exhibited no obvious phenotypic abnormalities. Deletion of ZIP3 did not alter zinc homeostasis at the molecular level as assessed by essential metal levels and the expression of zinc-responsive genes. In knockout mice stressed with a zinc-deficient diet during pregnancy or at weaning, a subtle increase in the sensitivity to abnormal morphogenesis of the embryo and to depletion of thymic pre-T cells, respectively, was noted. These results suggest that this protein plays an ancillary role in zinc homeostasis in mice.

Zip3 (Slc39a3) functions in zinc reuptake from the alveolar lumen in lactating mammary gland

The lactating mammary gland is composed of multiple cell types that tightly coordinate the accumulation, production, and secretion of milk components, including essential metals such as zinc (Zn). Our previous studies in animal and cell models implicated the Zn transporter Zip3 (Slc39a3) in mammary gland Zn acquisition. Herein, we investigated this hypothesis directly by utilizing Zip3-null mice. Our data verify that Zip3 is expressed in secretory mammary cells; however, Zip3 does not play a major role in Zn import from the maternal circulation. Importantly, the primary localization of Zip3 was associated with the luminal membrane of the secretory mammary cells. Consistent with this localization, Zn transfer studies using (65)Zn revealed that Zn retention in the secreted milk pool and milk Zn concentration was higher in Zip3-null compared with wild-type mice. Although total mammary gland Zn concentration was not altered, Zip3-null mice also had altered mammary tissue architecture, increased number of apoptotic cells, and reduced mammary gland weight implicating subtle changes in Zip3-mediated intracellular Zn pools in apoptosis regulation. Taken together, our data indicate that Zip3 does not participate in the acquisition of Zn from maternal circulation for secretion into milk but, in contrast, primarily plays a role in the reuptake and cellular retention of Zn in the mammary gland from the previously secreted milk pool, thus regulating cellular function.