Regulatory mechanisms of Slc39a4 (Zip4) and Slc39a5 (Zip5) in the adaptive response to zinc availability

Zinc is an essential micronutrient. Zinc deficiency results in severe dermatitis, immune dysfunction, diarrhea, mental retardation, and a failure to thrive. Zinc toxicity can result in irreversible nerve damage or severe pancreatitis. Thus, the ability to regulate proper zinc concentrations within the cell and throughout the body is essential for survival. Two families of zinc transporters mediate the proper levels of zinc within mammals through their cumulative actions. The Slc30a (ZnT) transporter family exports zinc from the cytosol; whereas, the Slc39a (Zrt-Irt-like protein, Zip) transporter family imports zinc into the cytosol. Mutations in several members of these transporter families give rise to distinct diseases. One such disease, acrodermatitis enteropathica, is a rare autosomal-recessive disease that is a result of inadequate zinc uptake from the diet due to mutations in the Slc39a4 (Zip4) gene. Loss of proper ZIP4 function ultimately results in death if untreated. Previous studies have shown that Zip4 and a closely related paralog, Zip5, are regulated by inverse mechanisms. The goals of this dissertation have been to determine the molecular mechanisms that regulate Zip4 and Zip5 in the adaptive response to zinc availability and how Zip4 impacts development. In this dissertation, I defend three specific aims. In the first specific aim, I evaluate the hypothesis that Zip4 is essential for the normal growth and development of mammals. To address this aim, a Zip4 knockout mouse model was employed. Loss of Zip4 resulted in embryonic lethality at the egg cylinder stage, prior to organogenesis. Heterozygosity had a negative association with eye, heart, and brain development and resulted in hypersensitivity to zinc deficiency. Excess zinc failed to rescue the lethal phenotype but ameliorated some of the heterozygous effects. In the second specific aim, I evaluate the hypothesis that Zip4 and Zip5 are post-transcriptionally regulated, inversely and dynamically in response to zinc availability. To address this aim, wild type mice were fed zinc adequate or zinc deficient diets. Some of the zinc deficient mice were repleted with zinc by oral gavage. Tissues known to express Zip4 and Zip5 were examined for expression of their mRNA levels, protein levels, and protein localization with time in response to changes in zinc availability. Zip4 and Zip5 had rapid and reciprocal regulation in response to zinc availability that was coordinated in multiple tissue types. Zip4 expression was regulated primarily by stability of the mRNA and protein: both accumulate during zinc deficiency; ZIP4 protein is rapidly internalized from the apical membrane of enterocytes and visceral endoderm then degraded in response to zinc repletion. Zip4 mRNA levels return to normal within 24 hours. Zip5 expression is apparently regulated by a translational stall mechanism during zinc deficiency. The Zip5 mRNA levels do not change with zinc availability and always remain polysome-associated; proteasome or lysosome inhibitor cocktails fail to restore ZIP5 protein levels. However, zinc repletion leads to the return of ZIP5 protein on the basolateral membrane of enterocytes, visceral endoderm, and pancreatic acinar cells. In the third specific aim, I evaluate the hypothesis that Zip5 is regulated by a rapid post-transcriptional mechanism mediated by the 3' untranslated region of the mRNA in response to zinc availability. To address this aim, in vivo and in vitro techniques were utilized. Several regulatory miRNAs were identified that are predicted to target the Zip5 mRNA in an accessible region of the well-conserved 3' UTR that is predicted to form a stable stem-loop structure. These miRNAs are polysome-associated in tissues known to regulate Zip5. These miRNAs are detected predominantly in precursor form, implying additional regulatory mechanisms. Further work is necessary to demonstrate a functional link between these predicted regulators and Zip5 expression. Altogether, this dissertation reveals that Zip4 and Zip5 are both regulated by intricate post-transcriptional mechanisms in response to zinc availability and that Zip4 is essential for development and proper zinc homeostasis.