Glen K. Andrews

Regulation of metallothionein gene expression by oxidative stress and metal ions.

The metallothioneins (MT) are small, cysteine-rich heavy metal-binding proteins which participate in an array of protective stress responses. Although a single essential function of MT has not been demonstrated, MT of higher eukaryotes evolved as a mechanism to regulate zinc levels and distribution within cells and organisms. These proteins can also protect against some toxic metals and oxidative stress-inducing agents. In mice, among the four known MT genes, the MT-I and -II genes are most widely expressed. Transcription of these genes is rapidly and dramatically up-regulated in response to zinc and cadmium, as well as in response to agents which cause oxidative stress and/or inflammation. The six zinc-finger metal-responsive transcription factor MTF-1 plays a central role in transcriptional activation of the MT-I gene in response to metals and oxidative stress. Mutation of the MTF-1 gene abolishes these responses, and MTF-1 is induced to bind to the metal response elements in proximal MT promoter in cells treated with zinc or during oxidative stress. The exact molecular mechanisms of action of MTF-1 are not fully understood. Our studies suggest that the DNA-binding activity of MTF-1 in vivo and in vitro is reversibly activated by zinc interactions with the zinc-finger domain. This reflects heterogeneity in the structure and function of the six zinc fingers. We hypothesize that MTF-1 functions as a sensor of free zinc pools in the cell. Changes in free zinc may occur in response to chemically diverse inducers. MTF-1 also exerts effects on MT-I gene transcription which are independent of a large increase in MTF-1 DNA-binding activity. For example, cadmium, which has little effect on the DNA-binding activity of MTF-1 in vivo or in vitro, is a more potent inducer of MT gene expression than is zinc. The basic helix-loop-helix-leucine zipper protein, USF (upstream stimulatory factor family), also plays a role in regulating transcription of the mouse MT-I gene in response to cadmium or H2O2. Expression of dominant negative USF-1 or deletion of its binding site from the proximal promoter attenuates induction of the mouse MT-I gene. USF apparently functions in this context by interacting with as yet unidentified proteins which bind to an antioxidant response element which overlaps the USF-binding site (USF/ARE). Interestingly, this composite element does not participate in the induction of MT-I gene transcription by zinc or redox-cycling quinones. Thus, regulation of the mouse MT-I gene by metals and oxidative stress involves multiple signaling pathways which depend on the species of metal ion and the nature of the oxidative stress.

The Transcription Factor MTF-1 Mediates Metal Regulation of the Mouse ZnT1 Gene

Metal regulation of the mouse zinc transporter (ZnT)-1 gene was examined in cultured cells and in the developing conceptus. Zinc or cadmium treatment of cell lines rapidly (3 h) and dramatically (about 12-fold) induced ZnT1 mRNA levels. In cells incubated in medium supplemented with Chelex-treated fetal bovine serum, to remove metal ions, levels of ZnT1 mRNA were reduced, and induction of this message in response to zinc or cadmium was accentuated (up to 31-fold induction). Changes in ZnT1 gene expression in these experiments paralleled those of metallothionein I (MT-I). Inhibition of RNA synthesis blocked metal induction of ZnT1 and MT-I mRNAs, whereas inhibition of protein synthesis did not. Metal response element-binding transcription factor (MTF)-1 mediates metal regulation of the metallothionein I gene. In vitroDNA-binding assays demonstrated that mouse MTF-1 can bind avidly to the two metal-response element sequences found in the ZnT1 promoter. Using mouse embryo fibroblasts with homozygous deletions of the MTF-1 gene, it was shown that this transcription factor is essential for basal as well as metal (zinc and cadmium) regulation of the ZnT1 gene in these cells. In vivo, ZnT1 mRNA was abundant in the midgestation visceral yolk sac and placenta. Dietary zinc deficiency during pregnancy down-regulated ZnT1 and MT-I mRNA levels (4–5-fold and >20-fold, respectively) in the visceral yolk sac, but had little effect on these mRNAs in the placenta. Homozygous knockout of the MTF-1 gene in transgenic mice also led to a 4–6-fold reduction in ZnT1 mRNA levels and a loss of MT-I mRNA in the visceral yolk sac. These results suggest that MTF-1 mediates the response to metal ions of both the ZnT1 and the MT-I genes the visceral yolk sac. Overall, these studies suggest that MTF-1 directly coordinates the regulation of genes involved in zinc homeostasis and protection against metal toxicity.

The heat shock response in HeLa cells is accompanied by elevated expression of the c-fos proto-oncogene.

Several known inducers of the heat shock response (heat stress, arsenite, and heavy metals) were shown to cause a significant elevation of c-fos mRNA in HeLa cells. Heat stress resulted in a time- and temperature-dependent prolonged elevation in the level of c-fos mRNA, which was accompanied by increased translation of c-fos protein and its appearance in the nucleus. Elevated expression of c-fos during heat stress was paralleled by induction of hsp 70 mRNA, while levels of c-myc and metallothionein mRNAs declined. Treatment of HeLa cells with arsenite or heavy metals also resulted in increased levels of hsp 70, as well as c-fos mRNA. Although elevated expression of c-fos was prevented by inhibitors of RNA synthesis, analysis of relative rates of gene transcription showed that during heat stress there was a negligible change in c-fos transcription. Therefore, the enhanced expression of c-fos during the heat shock response is likely to occur primarily through posttranscriptional processes. Cycloheximide was also shown to significantly increase the c-fos mRNA level in HeLa cells. There results are consistent with the observation that these inducers of the heat shock response, as well as cycloheximide, repress protein synthesis and suggest that the increase in the level of c-fos mRNA is caused by an inhibition of protein synthesis. This supports the hypothesis that c-fos mRNA is preferentially stabilized under conditions which induce the heat shock response, perhaps by decreased synthesis of a short-lived protein which regulates c-fos mRNA turnover.

The genetics of essential metal homeostasis during development

The essential metals copper, zinc, and iron play key roles in embryonic, fetal, and postnatal development in higher eukaryotes. Recent advances in our understanding of the molecules involved in the intricate control of the homeostasis of these metals and the availability of natural mutations and targeted mutations in many of the genes involved have allowed for elucidation of the diverse roles of these metals during development. Evidence suggests that the ability of the embryo to control the homeostasis of these metals becomes essential at the blastocyst stage and during early morphogenesis. However, these metals play unique roles throughout development and exert pleiotropic, metal‐specific, and often cell‐specific effects on morphogenesis, growth, and differentiation. Herein, we briefly review the major players known to be involved in the homeostasis of each of these essential metals and their known roles in development. genesis 46:214–228, 2008.

REVERSIBLE ACTIVATION OF MOUSE METAL RESPONSE ELEMENT-BINDING TRANSCRIPTION FACTOR 1 DNA BINDING INVOLVES ZINC INTERACTION WITH THE ZINC FINGER DOMAIN

The DNA-binding activity of the Zn finger protein metal response element-binding transcription factor 1 (MTF-1) was rapidly induced both in vivo in mouse Hepa cells, canine MDCK, and human HeLa cells after incubation in medium containing zinc and in vitro in whole-cell extracts to which zinc was added. Acquisition of DNA-binding capacity in the presence of free zinc was temperature and time dependent and did not occur at 4 degrees C. In contrast, activated MTF-1 binding to the metal response element occurred at 4 degrees C. After Zn activation, mouse MTF-1 binding activity was more sensitive to EDTA and was stabilized by DNA binding relative to the Zn finger transcription factor Sp1. After dilution of nuclear or whole-cell extracts from Zn-treated cells and incubation at 37 degrees C, mouse MTF-1 DNA-binding activity was no longer detected but could be completely reconstituted by the subsequent readdition of zinc. In vitro-synthesized, recombinant mouse MTF-1 displayed a similar, reversible temperature- and Zn-dependent activation of DNA-binding activity. Analysis of deletion mutants of recombinant MTF-1 suggests that the Zn finger domain is important for the Zn-dependent activation of DNA-binding capacity. Thus, mouse MTF-1 functions as a reversibly activated sensor of free zinc pools in the cell.

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.

The transcription factors MTF-1 and USF1 cooperate to regulate mouse metallothionein-I expression in response to the essential metal zinc in visceral endoderm cells during early development.

During early development of the mouse embryo, expression of the metallothionein‐I (MT‐I) gene is heightened specifically in the endoderm cells of the visceral yolk sac. The mechanisms of regulation of this cell‐specific pattern of expression of metallothionein‐I are unknown. However, it has recently been shown that MTF‐1, functioning as a metalloregulatory transcription factor, activates metallothionein genes in response to the essential metal zinc. In contrast with the metallothionein genes, MTF‐1 is essential for development; null mutant embryos die due to liver degeneration. We report here that MTF‐1 is absolutely essential for upregulation of MT‐I gene expression in visceral endoderm cells and that optimal expression also involves interactions of the basic helix–loop–helix upstream stimulatory factor‐1 (USF1) with an E‐box1‐containing sequence at −223 bp in the MT‐I promoter. Expression of MT‐I in visceral endoderm cells was dependent on maternal dietary zinc. Thus, the essential metal, zinc, apparently provides the signaling ligand that activates cell‐ specific MT‐I expression in visceral endoderm cells.

Defective epidermal growth factor gene expression in mice with polycystic kidney disease

The C57BL/6J-cpk mouse has an inheritable form of polycystic kidney disease similar to the autosomal recessive disorder seen in humans. Between approximately 1 and 3 weeks of age, affected cpk mice develop numerous large cysts in the collecting tubule segment of kidney nephrons. The present study examined the ontogeny of renal and submandibular gland prepro-epidermal growth factor (preproEGF) gene expression in the cpk mouse using Northern blot hybridization and immunohistochemistry. There was a virtual absence of renal preproEGF gene expression in cystic kidneys over the 3-week postnatal period, during which time renal preproEGF mRNA and proEGF/EGF protein normally reach significant levels. PreproEGF mRNA was expressed in salivary glands of cystic mice; however, this mRNA could not be further elevated with testosterone suggesting that there are abnormalities in the regulation of the preproEGF gene in the submandibular gland, as well as in the kidney. Since renal preproEGF expression during the early postnatal period occurs when collecting duct cysts form, it is possible that a deficiency in renal proEGF or EGF contributes to the rapid development of collecting duct cysts and the concomitant renal failure in the C57BL/6J-cpk cystic mouse.

Regulation and function of Zip4, the acrodermatitis enteropathica gene

The SLC39A (solute carrier 39A) [ZIP (Zrt-Irt-like protein)] family consists of 14 members which are thought to control zinc uptake into the cytoplasm. Among these, ZIP4 is known to be particularly important for zinc homoeostasis. Mutations in this gene cause acrodermatitis enteropathica, a rare recessive-lethal human genetic disorder. In the present paper, our studies of the regulation and function of the mouse Zip4 gene are briefly reviewed. Mouse Zip4 is expressed at highest levels in tissues involved in absorption of dietary or maternal zinc, and the gene and protein are dynamically regulated by multiple post-transcriptional mechanisms in response to zinc availability. ZIP4 accumulates at the apical surface of enterocytes and endoderm cells when zinc is deficient, because of increased stability of the mRNA and stabilization of the protein. In contrast, when zinc is replenished, the mRNA is destabilized and the protein is internalized and degraded rapidly. The critical importance of ZIP4 in zinc homoeostasis is revealed in mice with targeted deletions of this gene. Homozygous Zip4-knockout embryos die during early morphogenesis and heterozygous offspring are significantly underrepresented and display an array of developmental defects, including exencephalia, anophthalmia and severe growth retardation. Mice heterozygous for Zip4-knockout are hypersensitive to zinc deficiency, which suggests that humans heterozygous for this gene may also be very sensitive to zinc deficiency.

Novel proteolytic processing of the ectodomain of the zinc transporter ZIP4 (SLC39A4) during zinc deficiency is inhibited by acrodermatitis enteropathica mutations.

ABSTRACT The zinc transporter ZIP4 (SLC39A4) is mutated in humans with the rare, autosomal recessive genetic disease acrodermatitis enteropathica. In mice, this gene is essential during early embryonic development. ZIP4 is dynamically regulated by multiple posttranscriptional mechanisms, and studies of mouse ZIP4 reported herein reveal that the ectodomain, the extracellular amino-terminal half of the protein, is proteolytically removed during prolonged zinc deficiency while the remaining eight-transmembrane carboxyl-terminal half of the protein is accumulated on the plasma membrane as an abundant form of ZIP4. This novel ZIP4 processing occurs in vivo in the intestine and visceral endoderm, in mouse Hepa cells that express the endogenous Slc39a4 gene and in transfected MDCK and CaCo2 cells, but not HEK293 cells. In transfected MDCK and CaCo2 cells, the ectodomain accumulated and remained associated with membranes when zinc was deficient. ZIP4 cleavage was attenuated by inhibitors of endocytosis, which suggests that the processed protein is recycled back to the plasma membrane and that the ectodomain may be internalized. Ectodomain cleavage is inhibited by acrodermatitis enteropathica mutations near a predicted metalloproteinase cleavage site which is also essential for proper ectodomain cleavage, and overexpression of processed ZIP4 or ZIP4 with ectodomain truncations rendered the mouse Mt1 gene hypersensitive to zinc. These finding suggest that the processing of ZIP4 may represent a significant regulatory mechanism controlling its function.