These studies address research objectives of this RFA including: mechanisms of regulation of metallothionein (MT) gene expression, metal induction during development, and organs specificity in transgenic models. The overall objective of theses studies is to understand the regulation and functions of MT in reproduction and embryonic development in mammals. This information, in turn, is likely to provide important clues as to possible roles of MT in carcinogenesis. Mt plays a pivotal role in essential metal homeostasis and in protection from toxic metals. Zinc (Zn), copper (Cu), and cadmium (Cd) have dramatic effects on embryogenesis. Insufficient dietary Zn or Cu leads to abnormal development and reproductive failure, whereas Cd, is embryotoxic, teratogenic, and carcinogenic. Our studies on mouse embryogenesis establish that the MT genes are actively expressed in specific cell-types that surround the developing embryo from the time of implantation to near parturition, as well as in metal-treated reimplantation blastocysts and in fetal hepatocyte at midgestation. This suggests that MT has important functions during embryogenesis.
The specific aims of the proposed studies are to: 1) determine mechanisms regulating the cell-type specific expression of these genes in the reproductive tract and embryo, and 2) delineate the functions of these proteins during embryogenesis.
Specific aim 1 will be approached by; a) examining the effects of well-documented MT gene transcriptional regulators on MT gene expression in vivo and/or in vitro in mouse decidual cells, visceral endoderm, reimplantation embryos and fetal hepatocytes, b) determining the in vivo interactions of proteins with the MT-I gene promoter in specific cell-types, using genomic footprinting techniques, and c) ultimately mapping cis-acting promoter sequences involved in MT gene expression in these cell-types using transgenic mouse strains that express reporter genes under the control of MT-I promoter deletion mutants. A testable hypothesis is that tissue-specific enhancer elements exist in the mouse MT-I promoter. Delineation of MTs functional roles in vivo is hampered by a lack of strains of mice that exhibit defects (mutations) effecting MT gene expression. Thus, specific aim 2 will be approached by creating strains of transgenic mice that; a) display constitutively heightened, or b) misexpression of the MT genes. Transgenic mice that constitutively over- express MT, due to integration multiple copies of the intact mouse MT-I gene, are currently under study. Transgenic strains that over-express the MT gene in the pancreas, liver and uterus due to enhancer-driven transgene expression will be created. Gene expression will be determined at the level of mRNA by Norther, in situ and solution hybridization, and the reverse transcriptase-polymerase chain reaction. MT protein synthesis and accumulation will be determined by the Cd-heme assay, pulse-labeling western blotting, immunocytochemistry, and anion exchange chromatography. Transgenic mice will be analyzed for effects of aberrant MT gene expression on normal embryogenesis as well as on the embryos response to exogenous metals. A testable hypothesis is that misexpression of MT will alter metal homeostasis during pregnancy causing increased sensitivity to dietary Zn deficiency and reduced sensitivity to Cd toxicity.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA061262-06
Application #
6137524
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Yang, Shen K
Project Start
1993-12-15
Project End
2002-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
6
Fiscal Year
2000
Total Cost
$188,600
Indirect Cost
Name
University of Kansas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160
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Andrews, G K; Lee, D K; Ravindra, R et al. (2001) 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. EMBO J 20:1114-22
Andrews, G K (2001) Cellular zinc sensors: MTF-1 regulation of gene expression. Biometals 14:223-37
Langmade, S J; Ravindra, R; Daniels, P J et al. (2000) The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J Biol Chem 275:34803-9
Andrews, G K (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59:95-104
Chu, W A; Moehlenkamp, J D; Bittel, D et al. (1999) Cadmium-mediated activation of the metal response element in human neuroblastoma cells lacking functional metal response element-binding transcription factor-1. J Biol Chem 274:5279-84
Andrews, G K; Geiser, J (1999) Expression of the mouse metallothionein-I and -II genes provides a reproductive advantage during maternal dietary zinc deficiency. J Nutr 129:1643-8
Li, Q; Hu, N; Daggett, M A et al. (1998) Participation of upstream stimulator factor (USF) in cadmium-induction of the mouse metallothionein-I gene. Nucleic Acids Res 26:5182-9
Dalton, T P; Bittel, D; Andrews, G K (1997) Reversible activation of mouse metal response element-binding transcription factor 1 DNA binding involves zinc interaction with the zinc finger domain. Mol Cell Biol 17:2781-9
Dalton, T P; Li, Q; Bittel, D et al. (1996) Oxidative stress activates metal-responsive transcription factor-1 binding activity. Occupancy in vivo of metal response elements in the metallothionein-I gene promoter. J Biol Chem 271:26233-41

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