UNIVERSITY OF CINCINNATI Date:____05/12/2006_____ I, ____________ Lei He_____________________________________, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Environmental Health It is entitled: Identification and Characterization of Mammalian ZIP Transporters That Play Important Roles in Cadmium Uptake and Toxicity This work and its defense approved by: Chair: ___Daniel W. Nebert____________ ___Timothy P. Dalton__________ ___Iain Cartwright_____________ ___Li Jin______________________ _______________________________ Identification and Characterization of Mammalian ZIP Transporters That Play Important Roles in Cadmium Uptake and Toxicity A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati In partial fulfillment of the requirements for the degree of Doctorate of Philosophy (PhD) In the Department of Environmental Health of the College of Medicine 2006 By Lei He B.Med., Sun Yat-sen University of Medical Sciences, 1996 M.S., Sun Yat-sen University of Medical Sciences, 1999 Committee Chair: Daniel W. Nebert, MD Department of Environmental Health University of Cincinnati Abstract Cadmium (Cd2+, Cd) is a nonessential metal widely distributed in the environment. At the molecular level, little is known in vertebrates about how Cd is handled. Resistance to Cd-induced testicular damage is a recessive trait assigned to the mouse Cdm locus. We first narrowed the Cdm- containing region from 4.96 Mb to 880 kb, and found the Slc39a8 gene (encoding ZIP8 transporter) in this region. Expression of ZIP8 in cultured mouse fetal fibroblasts produced a >10-fold increase in the rate of intracellular Cd accumulation and 30-fold increase in Cd-induced cell death. Although the complete ZIP8 mRNA has no nucleotide differences between two sensitive and two resistant strains of mice, the ZIP8 mRNA was highly expressed in vascular endothelium of the testis in two sensitive inbred strains of mice but absent in these cells in two resistant strains. The Slc39a8 gene is therefore the Cdm locus. Cd uptake mediated by ZIP8 operates maximally at pH 7.5 and 37ºC, inhibited by cyanide, and - dependent on bicarbonate (HCO3 ). The Km for Cd uptake is 0.62 mM, when determined in Hank’s balanced salt solution (HBSS). Mn is the best competitive cation for Cd uptake, and the Km for Mn uptake is 2.2 mM in HBSS; thus, Mn is likely the physiological substrate. ZIP8 protein is glycosylated and is localized to the apical side of Madin-Darby canine kidney (MDCK) epithelial cells. Of 14 ZIP transporters identified in the human and mouse genome, ZIP14 is evolutionarily the one most related to ZIP8, having a similar number of amino acids, highest percent identity, and similar intron-exon structure. Two spliced ZIP14 transcripts, involving alternative exons 4, were found to translate into fully functional proteins, designated as ZIP14A and ZIP14B. Similar to the ZIP8 transporter, the ZIP14 proteins have a high affinity for Cd and Mn. ZIP14 proteins are heavily glycosylated and also localized to the apical side of MDCK cells. We believe that the ZIP8 and ZIP14 transporters are likely to play important roles in Cd uptake and disease in experimental animals, as well as humans. Acknowledgments I would like to thank members of my dissertation committee, Drs. Daniel W. Nebert, Timothy P. Dalton, Iain Cartwright, and Li Jin for all their suggestions and criticisms. I especially want to express my sincere gratitude to my advisor, Dr. Daniel Nebert, for the wonderful opportunity to work on this project. With your dedication, passion and discipline, you have been, and surely will continue to be, a role model scientist for me to look up to. I would also like to thank my lab mentor, good friend and big brother –Dr. Timothy Dalton –for giving me the biggest pressure and greatest help. Of all my academic accomplishments and personality improvements in the last 6 years, I can hardly think of anything that has been achieved without your help. Special thanks also go to the students from our lab: Bin Wang, Kuppuswami Girijashanker, and Jodie Reed. For sharing the good and bad times in our student lives, for valuable discussions and suggestions, and for the friendship and encouragement. I would also like to thank the many other lab members for various help. You have made the lab an enjoyable place to work. Finally, I would also like to thank my family members, especially my dad Wenchao He and my mom Fengzhu Wang, for their love, wisdom and unconditional support. I would also like to give special thanks to my wife, Yun Chen, for her love, encouragement, and providing me with balance to my life. Table of Contents List of Figures……………………………………………………………………………………3 Abbreviations……………………………………………………………………………………6 Chapter I Introduction to Cd Toxicity, Transport, and the Search for the Cdm Gene……….…………......8 Chapter II Identification of Mouse SLC39A8 as the Transporter Responsible for Cd-Induced Toxicity in the Testis Abstract…………………………………………………………………………………17 Introduction…………………..………………………………………………….……...17 Materials and Methods…………………………..………………………..….…………19 Results……………………………………………………………………..……………22 Discussion…………….……………………………..………………………………….27 Figure Legends……………………………………………………………..…………...31 Figures and Tables………………………………………. ……………….……………33 Chapter III Introduction to the ZIP Family of Transporters ………………………….…………………….40 Chapter IV ZIP8, Member of the Solute-Carrier-39 (SLC39) Metal Transporter Family: Characterization of Transporter Properties - 1 - Abstract………………………………………………………………………………….50 Introduction…………………..………………………………………………….………51 Materials and Methods…………………………..………………………..….…………52 Results……………………………………………………………………..……………56 Discussion…………….……………………………..………………………………….64 Figure Legends……………………………………………………………..…………...70 Figures and Tables………………………………………. ……………….……………73 Chapter V Cloning and Initial Characterization of Mouse ZIP14A and ZIP14B Transporters Abstract…………………………………………………………………………………81 Introduction…………………..………………………………………………….………82 Materials and Methods…………………………..………………………..….…………83 Results……………………………………………………………………..……………86 Discussion…………….……………………………..………………………………….91 Figure Legends……………………………………………………………..…………...95 Figures and Tables………………………………………. ……………….……………97 Chapter VI Conclusions and Speculations………………………………………………………………..…105 Chapter VII References (for all chapters)………………………………………………………………..….121 - 2 - List of Figures Chapter II. Figure 1 – Refinement of Cdm-gene-containing region 1A – Genetic map originally generated by Taylor et al, 1973 1B – Phenotype-genotype association studies with the recombinant inbred line BXD14/Ty 1C – SNP analysis over the 880-kb and three putatively functional genes Figure 2A – Northern blot of ZIP8 mRNA in rvZIP8 cells or rvLUC control cells 2B – Dose–response curves for Cd-induced cell death (MTT assay) 2C – Dose–response curves for Cd-induced cell death (LDH release assay) Figure 3 – Increased Cd uptake caused by membrane-localized ZIP8 109 3A – Time/dose-dependent CdCl2 uptake 3B – Western blot of ZIP8ha in microsomes (30 vs. 10 µg per lane). 3C – Western blot of ZIP8ha in cytosol (C) or microsomes (M) 3D – Localization of the ZIP8ha protein Figure 4 – Localization of ZIP8 mRNA in Mouse Testis 4A – Northern Analysis of Testicular ZIP8 mRNA 4B – In situ hybridization of ZIP8 mRNA in testis 4C – High-magnification bright-field ZIP8 in situ image of testicular capillaries Figure 5 – Three Alternative First Exons of Slc39a8 Chapter IV. - 3 - Figure 1 – Effects of temperature (A), KCN pretreatment (B), and pHout (C) on Cd uptake in cultured rvZIP8 and rvLUC cells Figure 2 – Metal cation competition for Cd uptake in rvZIP8 cells. Figure 3 – Comparison of the kinetics of Cd (A) and Mn (B) uptake in rvZIP8 cells. Figure 4 – Cell killing by 32-h exposure to Cd (A), Mn2+ (B), Hg2+ (C) or Zn2+ (D) in rvZIP8 versus rvLUC cells. Figure 5 – Effects of Na+ (A), K+ (B), or Cl– (C) substitution on Cd uptake - Figure 6A – Cd uptake as a function of HCO3 concentration in HBSS 6B – percent inhibition of Cd uptake by DIDS Figure 7 – Western immunoblot of control, ZIP4ha, and ZIP8ha proteins Figure 8 – Z-stack confocal microscopy of MDCK cells Table 1 – Composition of Hank’s Balanced Salt Solution (HBSS) Chapter V Figure 1A – Mouse ZIP14 alternatively spliced forms 1B – Phylogenetic tree of mammalian ZIP proteins 1C – Alignment of ZIP14 and ZIP8 proteins Figure 2 – Cd (A) and Mn (B) uptake by ZIP14 Figure 3 – Cd (A) and Mn (B) in vitro toxicity mediated by ZIP14 Figure 4 – Western blot of ZIP proteins in their over-expression cells Figure 5A – ZIP14 protein localization in non-polarized MFF cells 5B – ZIP14 protein localization in polarized MDCK cells Figure 6 – Tissue survey of ZIP8 and ZIP14 mRNAs - 4 - Table 1 – Comparison of Slc39a14 and Slc39a8 gene structure Chapter VI Figure 1 – Analysis of Celera SNPs in 170-kb region corresponding to the BAC Transgene - 5 - Abbreviations AEZ –acrodermatitis enteropathica, due to Zn deficiency B6 – C57BL/6J BANK1 – B-cell scaffold protein with ankyrin repeats 1 Cd – cadmium CITN – cadmium-induced testicular necrosis D2 – DBA/2J DCT – distal convoluted tubules DMEM – Dulbecco's Modified Eagle’s Medium DMT1 – divalent metal transporter-1 (official name: SLC11A2) EST – expressed sequence tag FBS – fetal bovine serum GFP – green fluorescent protein GSH – glutathione, reduced ha – hemagglutinin tag on C-terminus HBSS – Hank’s balanced salt solution - HCO3 – bicarbonate anion LZT – LIV-1 subfamily of ZIP Zn transporters