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
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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
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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.
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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
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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
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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
MDCK – Madin-Darby canine kidney cells
MFF – mouse fetal fibroblast
Mn – manganese
MT – metallothioneins-1 and -2
NBC1 – sodium-bicarbonate-1 cotransporter
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NCBI – National Center for Biotechnology Information rvZIP8, rvLUC – retrovirally-transformed MFFs expressing ZIP8 or firefly luciferase
Slc39 – solute carrier protein family 39 in mouse (SLC39 in human)
PBS – phosphate-buffered saline
PCT – proximal convoluted tubules
PNA – peanut agglutinin
ZIP – Zrt-, Irt-like protein
Zn – zinc
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Chapter I. Introduction to Cadmium Toxicity, Transport and the
Search for Cdm Gene
Cd and Its Toxicity to Mammals
Cadmium (Cd) is a group IIB metal that has an atomic weight of 112.41; it exists in the 0 or 2+ oxidation states. Cd is found naturally in the earth’s crust and is usually present in the environment as an inorganic salt. Heavy Cd usage began fairly recently with its large-scale application, dating from the 1940s (Stoeppler, 1991). Other human activities leading to the increase of Cd concentration in the environment include mining and the burning of fossil fuels (ATSDR, 1999).
Today its main uses include nickel–Cd battery manufacturing, pigments, and plastic stabilizers, whereas applications in alloys, solders and electroplating show a decreasing trend (Waisberg et al.,
2003).
Major occupational exposures to Cd occur in non-ferrous metal smelters, in the production and processing of Cd, its alloys and its compounds, and increasingly, in the recycling of electronic waste (Waisberg et al., 2003). In the general human population, cigarette smoke is by far the largest source of Cd exposure (Zalups and Ahmad, 2003). Each cigarette may contain from 1 to 2 mg of Cd, and 40–60% of the Cd in inhaled smoke generally can be taken into the systemic circulation
(ATSDR, 1999; Lewis et al., 1972; Elinder et al., 1976). For nonsmokers in the general population, ingestion of food contaminated with Cd is a major source of Cd exposure. Fish, organ meat
(especially from liver and kidney), and grain and cereal products usually contribute the greatest amount of Cd to the diet (WHO, 1992; ATSDR, 1999). The half-life of Cd in humans is estimated to be between 15 and 20 years (Jin et al., 1998).
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In humans and other mammals, Cd affects adversely a number of organs and tissues, including kidney, liver, lung, pancreas, testis, placenta, and bone, with the liver and kidney being two of the primary target organs (Zalups and Ahmad, 2003). The importance of Cd as an environmental contaminant is illustrated by the outbreak at the Jinzu River in Japan of a severe disease (Itai–Itai disease), characterized by severe pain, bone fractures, proteinuria and severe osteomalacia, which appeared mainly among women. This disease was caused by the ingestion of rice water contaminated with Cd originating in a mine slag. Overall, Cd has been ranked as high as seventh in the Top 20 Hazardous Substances Priority List by the Agency for Toxic Substances and Disease
Registry and the U.S. Environmental Protection Agency (Fay and Mumtaz, 1996).
Occupational exposure to Cd has been associated with cancer of the lung. The association of
Cd-induced cancer with the prostate, pancreas, and kidney has also been suggested, but the conclusion is quite controversial. Cd has been classified as a category I carcinogen (human carcinogen) of the USA (WHO, 1992; IARC, 1993; National Toxicology Program, 2000). In rodents, Cd induces tumors in various organs, such as adenocarcinomas of the lung after inhalation, prostate and pancreas tumors by subcutaneous injection, testicular tumors by oral exposure, and local tumors at various sites of injection (Waisberg et al., 2003). Cd is not a genotoxic carcinogen. It is essentially non-mutagenic in bacterial tests and only weakly mutagenic in mammalian cells in culture (IARC, 1993). However, it has been proven to be co-mutagenic in mammalian cell tests when combined with genotoxic agents, which may be due to its inhibitory effect on DNA repair
(Hartwig and Schwerdtle, 2002).
Molecular Mechanism of Cd Transport
In order to understand how Cd causes toxicity in the target cells, it is necessary to gain a thorough understanding of molecular events underlying Cd uptake and intracellular localization.
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There are polypeptides such as metallothionein (MT) and reduced glutathione (GSH) that bind and provide protection against Cd. Following intestinal (or lung) absorption, Cd binds to various polypeptides––such as MT, GSH, other thiol-proteins and plasma albumin. These Cd complexes in the blood, along with the Cd-binding to the surface of red blood cells, are the major forms for transporting Cd in the blood.
There are three hypotheses proposed for Cd uptake by epithelial cells (Bridges and Zalups,
2005). Cd may interact with membrane transporters for essential metals – such as Ca, Fe, and Zn, as a means to gain entry into target cells. Cd can also form linear II coordinate covalent complexes with certain sulfhydryl-containing biomolecules, such as GSH, cysteine (Cys), or homocysteine in certain compartments of the body (Rabenstein et al., 1983); subsequently, these complexes may be taken up by the transporters for endogenous amino acids, oligopeptides, organic anions, or organic cations. Receptor-mediated endocytosis of a Cd–protein complex, such as CdMT or Cd-albumin, also appears to be an important mechanism by which Cd is taken up by some epithelial cells.
Although numerous studies on Cd transport have been conducted, no direct and conclusive evidence for the molecular mechanism has been collected until very recently. For the first hypothesis, calcium channels and SLC11A2 (NRAMP2, DMT1), a proton-coupled divalent metal transporter with preference for iron, have been strongly implicated in Cd uptake and toxicity in mammals. In cultured pituitary cells, the major route of Cd influx is through voltage-gated calcium channels (Hinkle et al., 1987). In hepatocytes, where the calcium channels are of the receptor- operated type, about a third of the internalized Cd enters the cell through this type of channel
(Blazka and Shaikh, 1991). The role of calcium channels in cellular uptake of Cd has also been demonstrated in rat melanotrophs (Shibuya and Douglas, 1992), hepatic WRL-68 cells (Souza et al.,
1997), pheochromocytoma PC12 cells (Hinkle et al, 1994), and MDCK cells (Olivi and Bressler,
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2000). SLC11A2 has a preference for iron, but also transports Pb and Cd (Bressler et al., 2004). By performing SLC11A2 knockdown studies in human intestinal Caco-2 cells (Bannon et al., 2003), proton-dependent transport of Cd was shown. In vivo studies suggested that SLC11A2 participates in Cd2+ transport in enterocytes (Elisma and Jumarie, 2001; Tallkvist et al., 2001; Park et al., 2002) and distal tubular cells (Friedman and Gesek, 1994; Olivi et al., 2001). Consistent with these studies, Cd transport in Xenopus oocytes expressing human SLC11A2 produced Michaelis-Menten kinetics with a Km of 1.04 ± 0.13 mM (Okubo et al., 2003).
Cd-Induced Testicular Necrosis (CITN) and Cdm gene
Although numerous studies about Cd toxicity have been done, most of these studies were done at the tissue and organ level; we still do not know too much about the molecular mechanisms for Cd toxicity. Generally speaking, to dissect the molecular events and the genetic basis of a phenotype, studying a gene mutant (either induced by man or existing naturally) is a straightforward and effective approach. For example, the AHR (aryl hydrocarbon receptor) and ARNT (aryl hydrocarbon receptor nuclear translocator) pathway was first proposed in the study of the differential Cyp1a1 gene induction by benzo[a]pyrene between D2 and B6 mice; later on, this pathway was identified by the study of cell lines having induced genetic alterations in the Ahr and
Arnt genes (Nebert et al., 1993).
Nature has provided a fascinating genetic system as a foothold into identifying a gene involved in Cd toxicity. Rodents, given a single injection of Cd at a low dose that has no measurable effect on any other organ system, will exhibit profound testicular damage leading to permanent infertility
(Alsberg and Schwartze, 1919) (Parizek, 1957). The dose used was 10 mmoles of CdCl2 per kg body weight (1.8 mg/kg), subcutaneously administered (Parizek, 1957). The pathological changes include the edema and necrosis of the tubular and interstitial tissues, neutrophil infiltration,
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extravasation of red cells, and platelet plugging (Dalton et al., 2000b). Cd-induced toxicity to the seminiferous tubule endothelium appears to be common across all species having testes—mouse, frog, pigeon, rooster, armadillo and opossum (Chiquoine, 1964)
Most mouse strains are sensitive to CITN. But there are also some inbred strains of mice being resistant to CITN, even at lethal Cd doses. Resistant strains include: A/J, A/HeJ, BALB/cJ,
C3H/HeJ, C3HeB/FeJ, C57BL/6J, C57BL/10J, HRS/J, I/LnJ, LG/J, PRO/Re, SEC/1ReJ, WB/ReJ and WK/ReJ. Susceptible strains include: AKR/J, AU/SsJ, BDP/J, BUB/BnJ, CBA/J, CBA/CaJ,
CBA/H-T6J, C57BL/KsJ, C57BR/cdJ, C57L/J, C58/J, CE/J, DBA/1J, DBA/2J, LP/J, LT/ReJ,
MA/J, NZB/BnJ, P/J, PL/J, RF/J, ROP/Gn, RIII/2J, SEA/GnJ, SM/J, ST/bJ, SWR/J, WC/ReJ,
WH/ReJ and 129/J (Taylor et al., 1973). The strain distribution pattern reflects known relationships among inbred strains, with the exception of SJL/J. This study suggested that a single major gene might account for the difference in this CITN trait (Taylor et al., 1973).
By intercrossing and backcrossing various strains of sensitive mice with various strains of resistant mice, it was found that a single autosomal recessive gene, designated Cdm, determined the resistance to CITN (Taylor, 1976). Cdm was located in a 24.6 ± 8.8 cM region between the amylase
(Amy1) and variant-waddler (Va) genes on chromosome 12 (Taylor, 1976). Later, these two marker genes were shown to be actually on chromosome 3. Using B6 x D2 genetic crosses and B6 x D2 recombinant strain analysis (BXD RI), Cdm was further refined to a region flanked by two micro- satellite markers—D3mit110 and D3mit255, approximately 0.64 cM apart (Dalton et al., 2000b).
The fact that Cdm is limited in its distribution among inbred strains and is recessive suggests that Cdm is a mutant allele from wild-type, and that susceptibility is the normal state (Taylor et al.,
1973). This is supported by the observation that other mammals are sensitive to CITN (Parizek,
1960). Thus, resistance probably should be considered as “loss of function” (Dalton et al., 2000b).
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What might be the Cdm gene product? Proteins involved into the protection or sensitization of heavy metal toxicity can be broadly classified into five groups (Dalton et al., 2000): (a) direct sequestration of metal ions; (b) transcription factors that regulate metal ion sensitivity; (c) proteins participating in the cellular response to oxidative stress; (d) transporters for metal ions; (e) proteins in proteolytic pathways.
In Vivo Cd Uptake by Testicular Tissues
There are several reports indicating that sensitive inbred strains of mice take up more Cd in their testicular tissues than resistant strains of mice. Sensitive CBA/J and DBA/1J mice exhibited a greater uptake of 109Cd by the testis than resistant C57BL/6J and BALB/cJ mice (Lucis and Lucis,
1969). Sensitive DBA/2 mice exhibited twice the testicular uptake than resistant C3H and BALB/c mice (Hata et al., 1980). Sensitive 129/J mice accumulated 5-6 times Cd concentration in the testis than resistant A/J mice (Chellman et al., 1984). Sensitive DBA/2J, 129/J, and CD-1 mice had up to
3 times greater testicular Cd accumulation than the resistant C3H/HeJ and A/J mice (Shaikh et al.,
1993). One discordant report came from Meisler and coworkers (Meisler et al., 1979); they found no quantitative differences in Cd content—in the liver, kidney, and testis—between resistant
C57BL/6J and sensitive DBA/2J mouse strains. Most of these studies used a sub-toxic level of Cd, i.e., no testicular damage was induced at the dose level being used.
According to King, in the resistant mouse strain A/J, Cd transport was significantly attenuated in the testis, epididymis, and brain—tissues with vascular barriers, when compared to the sensitive mouse strain 129/J (King et al., 1998; King et al., 1999). He speculated that since only testis contains a fibrous tunica, which acts as a structural barrier to the dispersion of interstitial fluid, accumulation of Cd could lead to edema and finally necrosis in the testis. The transport system used by Cd in the 129/J testis was saturable and competitively inhibited by Zn. Consistently, the transport
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of 65Zn into testis and brain of resistant A/J mice was significantly reduced compared with sensitive
129/J mice (King et al., 2000). King’s experiments suggested that Cd was taken up by testis through an uptake mechanism which may normally transport Zn.
Blood-Testis Barrier and Its Implications
The concept of a blood-testis barrier was first conceived in the early 1900s. Studies on the “rete testis fluid” showed that it has a composition that is quite different from blood plasma or testicular lymph, both in ionic and organic constituents (Voglmayr et al., 1967; Setchell et al., 1969; Setchell and Waites, 1970). These differences could not be maintained if the fluid in the lumina of the seminiferous tubules could exchange readily with the fluids outside of the tubules. Different markers were infused into the blood stream. A wide range of entry rates into testicular lymph and rete or tubular fluid were found, depending mainly on lipid solubility, not on molecular size (Ploen and Setchell, 1992). Later studies showed that the endothelium of tesiticular vasculature is an important component of the blood-testis barrier. The testicular endothelium is continuous, nonfenestrated, and having long junctional profiles (Holash et al., 1993).
The existence of a blood-testis barrier implies that Cd cannot penetrate the testis readily. Thus, it is logical to speculate on the existence of some specialized transport system for Cd in the testicular vasculature, in order to explain the observed differential testicular uptake by different mouse inbred strains at the sub-toxic Cd level. And a secondary speculation that follows would be the more serious damaging effect of Cd at low concentrations to testicular vasculature than to the vasculature of other tissues. Experiments have supported the secondary hypothesis. In the untreated guinea-pig or rat, acriflavine produced weak staining of the nuclei of interstitial cells and cannot penetrate into the tubules. Following Cd administration, staining of all parts of the testis occurred (Johnson, 1969).
This was due to the damaging effect of the Cd to the endothelial cells of the vasculature, and the
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subsequent increase in their permeability. Testicular Blood flow in the testis and the initial segment of the caput of the epididymis sharply decreased 3 hrs later (Setchell and Waites, 1970). There were no obvious changes in the rest of the epididymis, skeletal muscle, or brain at the dose being used.
It may be of relevance to point out that acute Cd toxicity is also manifested in the cranial and spinal sensory ganglia (Gabbiani, 1966), and these ganglionic lesions resemble those in the testis with similar sequence of development and hemorrhagic nature (Gabbiani et al., 1967). Within the nervous system, the greatest accumulation was seen in peripheral ganglia, whereas the central nervous system (CNS) level is low. Within the CNS, most of the Cd was located in the hypophysis, meninges, choroid plexus and pineal gland, structures that do not have a blood-brain barrier
(Arvidson and Tjalve, 1985).
Pathogenesis of CITN and Vasculature Hypothesis
CITN was attributed to primary vascular injury (Gunn et al., 1963), a view which is supported by the early onset of increased permeability (Clegg and Carr, 1967; Gupta et al., 1967; Kormano and Suvanto, 1968; Johnson, 1969), elevated intratesticular pressure (Kormano and Suvanto, 1968) and decreased regional blood flow (Waites and Setchell, 1966; Setchell and Waites, 1970). Ultra- structural studies have correlated the increased vascular permeability with prominent pinocytosis of the testicular endothelium (Chiquoine, 1964) and with a widening of intercellular junctions through which extravasated marker substances readily pass (Clegg and Carr, 1967). It is also interesting to note that regenerated blood vessels in the regenerated testicular fibro-tissues lost its selective injurious vascular response to Cd (Gunn et al., 1966), although its implication is unclear.
From the above experimental evidence, we can delineate a tentative scheme for CITN pathogenesis in sensitive mice: endothelial cells take up more Cd à damage to endothelial cells à disruption of vasculature à testicular necrosis.
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In summary, testis is one of the most sensitive organs to Cd toxicity; some inbred mouse strains are resistant to Cd-induced testicular necrosis, and this resistance is predominantly determined by a single autosomal recessive gene, designated as Cdm. The identification of Cdm gene would surely reveal an important molecular mechanism for Cd toxicity in mammals.
Although many studies have been performed to understand the pathogenesis of CITN and the reason why some strains of mice are resistant to CITN, no conclusive mechanistic data have been collected. The nature of Cdm gene was still a mystery. By studying the CITN phenotype of BXD recombinant inbred strains, the Cdm-containing region was narrowed down to 0.64 cM on
Chromosome 3. There were still many genes in this region. Further efforts were needed to refine this Cdm-containing region.
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Chapter II. Identification of mouse SLC39A8 as the transporter
responsible for Cd-induced toxicity in the testis
Abstract:
Testicular necrosis is a sensitive endpoint for cadmium (Cd2+, Cd) toxicity across all species tested.
Resistance to Cd-induced testicular damage is a recessive trait assigned to the Cdm locus on mouse chromosome 3. We first narrowed the Cdm-gene-containing region to 880 kb. SNP analysis of this region from two sensitive and two resistant inbred strains demonstrated a 400-kb haplotype block consistent with the Cd-induced toxicity phenotype; in this region is the Slc39a8 gene encoding a member of the solute-carrier superfamily. Slc39a8 encodes SLC39A8 (ZIP8), whose homologs in plant and yeast are putative zinc (Zn2+, Zn) transporters. We show here that ZRT-, IRT-like protein
(ZIP)8 expression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of intracellular Cd influx and accumulation and 30-fold increase in sensitivity to Cd-induced cell death.
The complete ZIP8 mRNA and intron-exon splice junctions have no nucleotide differences between two sensitive and two resistant strains of mice; by using situ hybridization, we found that ZIP8 mRNA is prominent in the vascular endothelial cells of the testis of the sensitive strains of mice but absent in these cells of resistant strains. Slc39a8 is therefore the Cdm gene, defining sensitivity to
Cd toxicity specifically in vascular endothelial cells of the testis.
(Introduction)
Cd is a toxic and carcinogenic nonessential metal (Jarup, 2003), which can enter the body through the intestine, skin, and lung and accumulates in the kidney (Swiergosz-Kowalewska, 2001;
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Takebayashi et al., 2003) . The level of Cd in the environment has risen with advances in industrialization, and the role of Cd in human disease is of increasing concern. The mechanisms of
Cd toxicity are poorly understood, although it is known that Cd exerts its effects intracellularly, and there are polypeptides such as metallothionein (Klaassen and Liu, 1998) and reduced glutathione
(Singhal et al., 1987) that bind Cd and afford protection. The subcellular events by which Cd is taken up by cells or removed from cells remain obscure, although such knowledge could provide potential therapeutic targets for protection or intervention against Cd toxicity. Several proteins transport Cd into bacteria, yeast, plants, and mammalian cells in culture (Williams et al., 2000;
Himeno et al., 2002; Hall and Williams, 2003; Morgan and DeCoursey, 2003; Thevenod, 2003;
Bressler et al., 2004), but their specific roles in causing toxicity are unclear; these studies underscore the difficulties in extrapolating from observations in cell culture to the intact animal.
Nature has provided a fascinating genetic system as a foothold into identifying a gene involved in Cd toxicity. It is known that Cd-induced testicular necrosis is common across all animal species having testes: rodents, opossum, armadillos, frogs, pigeons, roosters, and fish (Alsberg and
Schwartze, 1919; Parizek and Zahor, 1956; Parizek, 1957; Chiquoine, 1964; Sangalang and
O'Halloran, 1972). Cellular events that precede Cd-induced testicular toxicity indicate that vascular endothelial cell injury is the earliest and, perhaps, the causative event (Gunn et al., 1963; Chiquoine,
1964; Waites and Setchell, 1966; Clegg and Carr, 1967; Gupta et al., 1967; Johnson, 1969; Setchell and Waites, 1970; Schlaepfer, 1971).
Some inbred mouse strains are resistant to Cd-induced testicular toxicity (Lucis and Lucis,
1969). The resistance phenotype segregates largely as an autosomal-recessive Mendelian trait, and the gene responsible for the trait was named Cdm (Taylor et al., 1973). The wild-type Cdm allele thus confers testicular sensitivity to Cd. The Cdm gene was mapped to a 24-centiMorgan (cM)
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segment (Taylor, 1976) (Fig. 1a) between amylase-1 (Amy1) and varitint-waddler (Va) on mouse
Chr 3 (Bonhomme et al., 1979). Phenotyping 26 BXD/Ty recombinant inbred lines and using quantitative histology to assess testicular necrosis, our lab refined the Cdm gene locus from >24 cM to 0.64 cM (Dalton et al., 2000b). In this study, we now have identified the Cdm gene as a member of the solute carrier Slc39 gene family.
Materials and Methods:
Animals. All mouse experiments were conducted in accordance with the National Institute of
Health standards for the care and use of experimental animals and the University of Cincinnati
Medical Center Institutional Animal Care and Use Committee. C57BL/6J (B6), DBA/2J (D2),
129S6/SvEvTac (129S6), and A/J inbred strains and the BXD14/Ty recombinant inbred line were purchased from The Jackson Laboratory.
Mapping the Cdm Locus. DNA from the above-described mice was prepared by using standard methods. Potential polymorphic microsatellite markers were identified by PCR by choosing primer sets adjacent to d(CA) repeats, generally >20 repeats in length. PCR was conducted, and amplicons from B6 DNA were compared with those from D2 DNA. PCR products showing
>5% difference by agarose gel electrophoresis were chosen for analysis. Recently discovered microsatellite markers and SNP sites (Fig. 1b) were amplified by using primer sets shown in Table 1, which is published as supporting information on the PNAS web site.
For fine-mapping the chromosomal crossover positions in B6D2F1 x B6 backcross offspring, we used several SNPs. The positions of these SNPs are shown in the context of 20 bp of contiguous sequence in Table 2, which is published as supporting information on the PNAS web site. SNPs
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analysis was conducted by using SNPs documented in the Celera Mouse Genome Database
(www.celeradiscoverysystem.com).
Treatment of the Mice. Testicular sensitivity to Cd was assessed as described in ref.(Dalton et al., 2000b).
Cloning of the Solute-Carrier (SLC)39A8 (ZIP8) cDNA. Oligo-dT-primed reverse transcription was carried out on B6 and D2 mouse total testicular RNA. Primers for amplification began at the start codon and ended at the stop codon; a consensus Kozak sequence at the start site was included for efficient expression. Restriction sites were added at the 5' (BamHI) and 3' (ClaI) ends for cloning into the pRevTRE vector (Invitrogen). A mutant ZRT-, IRT-like protein (ZIP)8
(ZIP8m) was generated by using a 5' primer with a single-base deletion in the third codon of the
ZIP8 cDNA. ZIP8 with a C-terminal hemagglutinin (HA) tag was generated (ZIP8ha) by using PCR and a 3' primer in which the termination codon was replaced with the coding sequence of an HA tag, followed by a termination codon.
Delivery of ZIP8 cDNA into Mouse Fetal Fibroblasts Tet-Off Cells. The cDNAs described below were inserted into the pRevTet-off vector (modified from the Invitrogen vector by replacing
G418 resistance with puromycin resistance; Bergwitz et al., 2000), which were used to generate retrovirus and infect immortalized mouse fetal fibroblasts (Solis et al., 2002) that express a Tet-off
Tet receptor. Cells were selected for resistance to puromycin (3 µg/ml). Cells were infected with retrovirus (rv)-encoding control luciferase (LUC), ZIP8, ZIP8m, or ZIP8ha to generate the rvLUC, rvZIP8, rvZIP8m, and ZIP8ha cell lines, respectively.
Northern Blot Hybridization. ZIP8 mRNA levels were measured by standard analysis (Dalton et al., 2000a).
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Western Immunoblot Analysis. The rvZIP8ha cells and rvLUC control cells were harvested in
PBS and homogenized (100 strokes with a tight-fitting Dounce homogenizer) in 10 mM Tris, pH
7.4/10 mM KCl9491 mM EDTA containing phenylmethylsulfonyl fluoride, leupeptin, and aprotinin.
Homogenates were centrifuged at 500 x g for 10 min and then 20,000 x g for 20 min. This supernatant was centrifuged at 100,000 x g for 30 min to generate a soluble cytosolic fraction, and the pellet was suspended by pipetting in homogenization buffer to generate a membrane fraction.
Proteins were quantified by using the BCA protein assay (Pierce). Proteins were denatured, run on
SDS/PAGE gels, transferred to nitrocellulose, and blotted (Dalton et al., 2000a). A rabbit affinity- purified polyvalent anti-HA antibody (Bethyl Laboratories; Montgomery, TX) was used at 1/10,000 dilution.
Immunohistochemical Analysis. The rvZIP8ha and rvLUC control cells were grown on fibronectin-coated cover slips, fixed, blocked, and then reacted with the anti-HA antibody
(described above; 1/2,000 dilution) and secondary FITC-conjugated goat anti-rabbit antibody (Alexa
Fluor 488, Molecular Probes) as described by (Robison et al., 2004). Images were recorded by confocal microscopy (Zeiss LSM510).
Measuring Cd Uptake. Cd uptake was performed (Jumarie et al., 1997) by control cell lines with 109Cd [3.64 mCi/mg (1 Ci = 37 GBq) in 0.5 M HCl; NEZ058; PerkinElmer].
Measuring Cd Toxicity. Cd toxicity was assessed in cell lines after a 32-h treatment with the indicated Cd concentration. Cell viability was determined as the cleavage of 3-(4,5-dimethlythiazol-
2-yl)-2,5-diphenyl tetrasodium bromide or lactate dehydrogenase release, both according to the manufacturer's protocol (Promega).
In Situ Hybridization. Templates for cRNA probes included a portion of the ZIP domain unique to ZIP8 and were generated by PCR with the primers: P081, 5'-
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AATTAACCCTCACTAAAGGGGGATCCGCTATGCCAACCCCGCTG-3'; and P082, 5'-
GTAATACGACTCACTATAGGGCATCGATGCAAGATCACAAAGTCCCCT-3'.
PCR products contained a 5' T3 polymerase promoter and 3' T7 polymerase promoter for generation of sense and antisense probes, respectively. We prepared the 35S-labeled single-stranded
RNA probes (2 x 109 cpm/µg) by using uridine 5'-( -[35S]thio)triphosphate (800 Ci/mmol) and T3 and T7 polymerases. Tissues were fixed, sectioned, and hybridized (Qian et al., 2003). After light photomicroscopy, coverslips were removed with xylene; residual emulsion was digested with 1% sodium hydroxide. After stepwise washes (water, Kodac fixer, water, and PBS), indirect immunofluorescence was performed by using anti-CD31 (Pharmingen), a biotinylated secondary antibody, and a fluorophore-tagged label (Alexa Fluor-488 streptavidin; catalog no. S11223
[GenBank] ).
Statistical Analysis. Statistical significance between groups was determined by way of analysis of variance for means and SD (95% confidence intervals) between each group by statistical linear models and Student's t test. All assays were performed in duplicate or triplicate and repeated at least
twice. Statistical analyses were performed with the use of SAS statistical software (SAS Institute,
Cary, NC). The determinations of Km and Vmax values for ZIP8 and ED50 values for Cd toxicity were
determined by using SIGMA PLOT (developed by Jandel Scientific; purchased by SPSS, Chicago; and sold by Systat Software, Point Richmond, CA).
Experimental Results:
Refinement from 4.96 Mb to 880 kb. The 0.64-cM Cdm-gene-containing region was determined (Dalton et al., 2000b) to be the 4.96-Mb segment between D3Mit110 and D3Mit255
(Fig. 1b). By using newly discovered polymorphic microsatellite markers, this region was decreased
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further to a 2.37-Mb segment defined by markers M12–17 and M10–26, in which D2 (sensitive) markers are flanked by B6 (resistant) markers in the Cd-sensitive BXD14/Ty recombinant inbred
line. DNA from 1,164 B6D2F1 x B6 backcross offspring was then analyzed by multiplex PCR, and nine informative previously uncharacterized recombinants further narrowed the Cdm-containing region to 880 kb, residing between S901 and M10–26 (Fig. 1b). This 880-kb segment contains two pseudogenes and three putatively functional genes: Bank1, encoding a B cell scaffold protein and signaling molecule with ankyrin repeats (Yokoyama et al., 2002); mcg22364 having no known function; and Slc39a8, a member of the SLC39 family (Eide, 2004) of the solute-carrier gene superfamily (Hediger et al., 2004). We resequenced all exons (including 5' and 3' untranslated regions) and all intron-exon splice junctions (at least 30 bp) of these three genes, comparing two Cd- sensitive inbred strains (D2 and 129S6) with two Cd-resistant inbred strains (B6 and A/J). Although only two nonsynonymous-coding SNPs in the Bank1 gene were detected, neither was in accord with the strain distribution pattern for Cd-induced testicular toxicity and, thus, neither is a candidate for the Cd toxicity trait.
Analysis of all SNPs (from the Celera Mouse Genome Database) in the two Cd-sensitive and two Cd-resistant inbred strains between S901 and M10–26 revealed a 400-kb haplotype block predictive of phenotype (Fig. 1c), suggesting an ancestral relationship between the sensitive and resistant strains, and further refined the Cdm-gene-containing region; also, this analysis eliminated
Bank1 as a candidate gene.
Two genes thus remained as Cdm candidates. The mcg22364 gene encodes an 87-residue protein, based on its longest ORF. Neither the gene nor its protein shares homology with any other gene or protein in the National Center for Biotechnology Information database. This gene is hypothetical, based on only two ESTs.
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Association of Cd Toxicity with the SLC39A8 Transporter. The remaining gene, Slc39a8, is one of 14 members so far identified in the mouse Slc39 family of metal-ion transporters; 15 SLC39 genes exist in the human genome (Eide, 2004). SLC39 genes are members of the ZIP family, best known for ZRT1 and ZRT2, the major Zn2+ uptake transporters in Saccharomyces cerevisiae, and
IRT1, the major iron transporter in Arabidopsis thaliana. ZIP proteins have been described (Eide,
2004) as transporters of Zn2+, Fe2+, and Mn2+. In general, ZIP proteins transport metal ions from outside the cell, or they are transported from intracellular organelles into the cytoplasm.
We found that ZIP8 mRNA is expressed in rvZIP8 cells but not in the control rvLUC cells (Fig.
2a). Cd was toxic to the rvZIP8m cells and rvLUC cells, both negative controls, with an ED50 for cell death of 22 µM, as seen by both the 3-(4,5-dimethlythiazol-2-yl)-2,5-diphenyl tetrasodium bromide (Fig. 2b) and lactate dehydrogenase (Fig. 2c) assays. The same was true for the parent mouse fetal fibroblast cell line (data not shown). On the other hand, rvZIP8 cells and rvZIP8ha cells
were sensitized to Cd, with an ED50 of 0.69 µM. We conclude that ZIP8 is a putative metal transporter whose expression sensitizes cells to Cd toxicity.
Membrane-Associated ZIP8 Stimulates Cd Uptake. The rate of intracellular Cd uptake was linear for at least 1 h with <25% of Cd internalized. At 0.25 and 1 µM Cd, rvZIP8 cells accumulated
Cd at 60 and 180 pmol/min per mg protein, respectively (Fig. 3a), compared with 5.3 and 12.4 pmol/min per mg protein, respectively, in rvLUC control cells. Because of the striking increase in
Cd uptake by ZIP8, we determined that Cd transport by ZIP8 is temperature-sensitive and saturable
(data not shown). The apparent Km for ZIP8-mediated transport of Cd is 8.4 ± 0.84 µM with a Vmax of 204 ± 6.6 pmol/min per mg protein. These studies were conducted in the presence of 10% FBS, meaning that transport can be directly compared to toxicity studies; serum constituents are known to
bind Cd such that its apparent Km is likely to be a considerable overestimation of the actual Km value.
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Is ZIP8 located in the plasma membrane? Western blot analysis of rvZIP8ha cells showed that
ZIP8 is detected as a tightly running doublet, migrating at 55 kDa (Fig. 3b). After overexposure, the ZIP8ha protein was seen in the membranes but not detected in cytosol (Fig. 3c). In addition to the abundant 55-kDa form of ZIP8ha, a slower-migrating 86-kDa form is detected; the nature of this band is not known. We had used rvZIP8ha cells to determine the cellular localization of ZIP8; the rvZIP8ha cells are sensitized to Cd to the same degree as rvZIP8 cells (Fig. 2b), suggesting that addition of the C-terminal HA tag does not affect transporter function. ZIP8ha is strictly associated with the plasma membrane (Fig. 3d). When rvZIP8ha cells were made permeable by using detergents, ZIP8ha was detected in membranes throughout the cell (data not shown).
ZIP8 mRNA and in Situ Hybridization Analysis. All Slc39a8 exons and splice junctions revealed no differences between two sensitive (D2 and 129S6) and two resistant (B6 and A/J) mouse strains; this finding suggests that a strain-specific difference exists in the testicular accumulation of ZIP8 mRNA, rather than a mutated or absent ZIP8 protein. Northern blot analysis of poly(A+) RNA from the testis of these four inbred strains showed no difference in the absolute levels of ZIP8 mRNA between the sensitive and resistant mouse strains (Fig. 4a). Real-time PCR of the whole testis also revealed no significant differences in ZIP8 mRNA levels among the two sensitive and two resistant strains (data not shown).
In situ hybridization was therefore performed. Analysis of the silver-grain distribution shows that ZIP8 was expressed similarly in the seminiferous tubules of all four inbred strains, with most silver grains over Sertoli cells (Fig. 4b). Sense-strand controls showed a diffuse nonspecific pattern of hybridization. Importantly, the two sensitive strains showed robust ZIP8 hybridization consistent with vascular endothelial cells of the testis; in contrast, this hybridization pattern was absent in the two resistant strains (Fig. 4b). High magnification bright-field photos of the interstitium showed the
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typical difference in silver-grain density between resistant and sensitive strains (Fig. 4c Upper); this analysis shows a sensitive-strain-specific accumulation of ZIP8 hybridization over testicular vascular endothelial cells. As further proof of endothelial localization, a capillary from the sensitive
129S6 strain with high silver-grain density was photographed, stripped of photographic emulsion, and reacted with an endothelial-cell-specific antibody (Fig. 4c Lower). High silver-grain density can be observed to colocalize with endothelial cell-specific staining. These data are consistent with endothelial cell ZIP8 mRNA accumulation in sensitive strains of mice, consistent with previous studies that vasculature damage is an early event of testicular necrosis. In accord with these findings, loss of vascular endothelial ZIP8 mRNA expression in the resistant inbred strains protects the testis from Cd toxicity.
Northern blot analysis of ZIP8 mRNA in total RNA (data not shown) showed an order of concentration of lung > kidney > liver = testis. A similar pattern has been demonstrated for ZIP8 in human tissues (Begum et al., 2002). ESTs for ZIP8 are ubiquitous, furthermore, found in >30 tissues and cell types (www.ncbi.nlm.nih.gov). Hence, possible participation of ZIP8 in Cd toxicity could be widespread. Examining ZIP8 expression in other tissues by in situ hybridization, we found that
ZIP8 mRNA was detected in lung, kidney, liver, lung, and intestine without differences in cell-type- specific expression between the two sensitive and two resistant strains of mice (data not shown).
Slc39a8 Gene Structure and Alternative Splicing. What is the mechanistic basis for the difference in accumulation of ZIP8 mRNA in vascular endothelial cells of the testis between these inbred strains? Based on ESTs from various tissues ("sequence information" entry in NCBI
UniGene link of Mm.30239 and Hs.284205), the mouse Slc39a8 and human SLC39A8 genes both have nine exons; introns 2–8 and exons 2–9 span >66 kb and >83 kb in mouse and human, respectively (Table 3, which is published as supporting information on the PNAS web site). Both
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species have three exons 1 as the result of alternative promoter usage and splicing, and several distinct exons 9 were caused by the use of different poly(A+) sites.
If the mouse Slc39a8 exons 1 (Fig. 5, which is published as supporting information on the
PNAS web site) are driven by independent promoters or enhancers, perhaps one of these promoters/enhancers might specify testicular endothelial-specific expression of ZIP8 mRNA. Would the transcript driven by this promoter be specifically absent from testicular RNA of Cd-resistant mice? Using real-time PCR to determine the levels of the transcripts initiated from each promoter, we could not detect the mRNA-containing exon 1c in testis; transcripts initiated at exons 1b and 1a were measurable but did not differ in amount between the sensitive and resistant strains (data not shown). Thus, neither the promoter that initiates at exon 1a nor exon 1b is specific to the testis.
Discussion:
Cd is a nonessential metal. Therefore, ZIP8 is not a Cd transporter per se but likely has evolved to transport one or more essential metals. Other members of the SLC39 family have been shown to transport divalent Zn, Fe, and Mn (Eide, 2004). During the course of uptake studies, we found that
the transport of 0.25 µM CdCl2 was 50% inhibited by divalent Zn and Mn at concentrations of 13
µM and 2.9 µM, respectively (data not shown). These studies suggest that ZIP8 is more selective for transport of Mn than Zn, although the cation transported by ZIP8 in cells cannot be predicted based solely on inhibitor selectivity.
The data in this article strongly suggest that Slc39a8 is the Cdm gene and that ZIP8, the transporter product of the mouse Slc39a8 gene, functions normally for Mn+2, and perhaps Zn+2, ions.
Cd+2 presumably participates as an opportunistic hitchhiker, being transported inadvertently into the vascular endothelial cells of the testis, resulting in increased cellular accumulation and toxicity.
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There exists a haplotype block, shared in a phenotype-specific manner among at least the four strains studied herein and encompassing Slc39a8 (Fig. 1c). As a result, a majority of SNPs in and around the Slc39a8 gene are shared by sensitive or resistant strains, respectively. This conclusion limits our ability to eliminate nonfunctional SNPs. By alignment of the SLC39A8 gene and surrounding sequences in humans and rats (both presumed to be Cd-sensitive) and Cd-sensitive mouse strains, versus Cd-resistant mouse strains, there are less than a dozen "candidate SNPs" within 10 kb of the 5' flanking region and the three largest introns (data not shown); however, because the expression difference between sensitive and resistant mouse strains is apparently limited to testicular vascular endothelial cells, determining perhaps a single SNP that results in a change in expression will likely require analysis in transgenic mice. Nonetheless, our data strongly implicate a difference in organ- and cell-type-specific Slc39a8 transcription as the mechanistic basis of resistance to Cd-induced testicular toxicity, but whether this result is due to an altered enhancer, locus-control region, or other DNA element remains to be determined.
Mammalian SLC39A8 is a largely uncharacterized gene. During a screen of innate immune activation of monocytes (Begum et al., 2002), human ZIP8 mRNA levels increased after treatment of monocytes with live and heat-killed Mycobacterium bovis bacillus Calmette–Guérin cell-wall lipopolysaccaride and inflammatory cytokines such as type- TNF.
The very ancient SLC superfamily includes genes encoding passive transporters, ion-coupled transporters, and exchangers. Currently, there are 43 families with 298 putatively functional transporter genes (Hediger et al., 2004). Metals are known to be transported by five families: SLC11 proton-coupled metal-ion transporter (Mackenzie and Hediger, 2004), SLC30 Zn effluxor (Palmiter and Huang, 2004), SLC31 copper transporter (Petris, 2004), SLC39 metal-ion transporter (Eide,
2004), and the SLC41 MgtE-like magnesium transporter (Hediger et al., 2004). Except for the
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SLC30 family of effluxors (formerly known as the cation diffusion facilitator family), transporters in the other four families pump metal ions into the cell.
Cd uptake and efflux, and the inhibition of metal transport by Cd, are well characterized in plants, yeast, and mollusk (Guerinot, 2000; Williams et al., 2000; Hall and Williams, 2003). In mammalian-cultured cells, many studies of Cd transport and inhibition of divalent cation transport by Cd have shown Cd uptake and toxicity (Gaither and Eide, 2000; Gaither and Eide, 2001; Bannon et al., 2003; Okubo et al., 2003; Thevenod, 2003), but there were no studies correlating Cd uptake with toxicity in any organ or specific cell type of any intact vertebrate.
Is ZIP8 important in Cd uptake and toxicity in mammalian organ systems aside from testis? As we have shown, the inbred strains used in this study do not display differences in ZIP8 mRNA accumulation in tissues examined except the endothelial cell of the testis. ZIP8 is present in a variety of tissues, including lungs and kidneys, two important target organs for Cd toxicity. Testicular endothelial cells are a unique population, involved in maintaining a blood–testis barrier with relatively impermeable tight junctions (Kamimura et al., 2002); this barrier is structurally different from other vascular endothelial cells, even when compared with the blood–brain barrier. It is our understanding that testicular vascular endothelial cells represent a very special endothelial cell population and that no good in vitro model for these cells exists.
Our observation of vascular endothelial cell toxicity might also give important insights into the molecular mechanisms of other types of heavy metal-associated human diseases. Epidemiological studies suggest Cd exposure can lead to testicular tumors in humans (Waalkes, 2003; Jarup, 2003);
Cd-induced cancer of the rodent testis has been shown experimentally (IARC, 1993; Waalkes,
2003). In humans, chronic exposure to Cd leads to renal and pulmonary toxicity, possibly osteoporosis, and "itai-itai" disease (IARC, 1993; Waalkes, 2003; Takebayashi et al., 2003).
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Interindividual variations in susceptibility to Cd toxicity might exist among people from the same area, with presumed similar amounts of exposure to Cd from the soil (Elinder et al., 1985;
Takebayashi et al., 2003); these data suggest that allelic differences in one or more human genes might be involved in resistance to Cd toxicity.
In conclusion, we have shown here that the absence of Slc39a8 expression in vascular endothelial cells of certain inbred mouse strains is associated with resistance to Cd-induced testicular toxicity, a phenotype ascribed to the Cdm gene. Although numerous studies of Cd uptake and toxicity have been performed in bacteria, yeast, plants, invertebrates, and mammalian cell culture, the gene Slc39a8 demonstrates Cd toxicity in an intact vertebrate. Slc39a8 is expressed in a variety of tissues, but its differential expression among inbred mouse strains is apparently specific to the vasculature of the testis.
Acknowledgements:
The preceding chapter was published in Proceedings of National Academy of Sciences USA
2005 Mar 1; 02(9):3401-6. (http://www.pnas.org/cgi/reprint/102/9/3401). The authors are Lei He,
Timothy P. Dalton, Bin Wang, Marian L. Miller, Li Jin, Keith F. Stringer, Xiaoqing Chang, C.
Stuart Baxter, and Daniel W. Nebert. T.P.D. and L.H. contributed equally to this work.
We thank our colleagues, especially David P. Witte and Alvaro Puga, for valuable discussions, technical advice, and a critical reading of the manuscript; and Jingchun Luo, Joanna Watson and Ge
Zhang (DNA studies), Stacey Andringa (histology), and Meredith Farmer, Chris Woods, and Pam
Groen (in situ hybridization) for technical assistance. This work was supported in part by National
Institutes of Health Grants R01 ES10416 (to D.W.N.) and P30 ES06096 (to T.P.D., M.L.M., L.J., and D.W.N.).
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Figure Legends:
Figure 1. Scheme showing how the Cdm-gene-containing region was refined from >24 cM on mouse Chr 3 to 4.96 Mb, 2.37 mM, and 880 kb containing three putatively functional genes. (a)
Genetic map originally generated by Taylor et al. (Taylor et al., 1973). Varitint waddler (28) has now become the mucolipin 3 gene (Mcoln3) (Zhu et al., 2003). (b) Phenotype-genotype association studies with the recombinant inbred line BXD14/Ty (b, B6; d, D2 allele) showing that a double crossover occurred between M12-7 and M10-26 (arrows above) and then nine recombinants derived from the (B6D2)F1 x B6 backcross further refined the Cdm locus to a region between S901 and
M10-26 (arrows below). Gray circled genotypes are recombinants that ultimately define the 880-kb segment containing Cdm. (c) SNP analysis over the 880-kb segment showing "the fraction of positive SNP signatures" occurring in 40-kb intervals. "Positive" denotes those SNPs in D2 and
129S6 (Cd-sensitive strains) that differ from that in B6 and A/J (Cd-resistant strains) divided by the total number of SNPs in 40-kb intervals.
Figure 2. Cd toxicity in mouse fetal fibroblasts that express ZIP8 cDNA. (a) Northern blot of
ZIP8 mRNA in rvZIP8 cells or rvLUC control cells. Total RNA was size-separated, and the blots were hybridized with cRNA probes for the indicated mRNA. ACTB, -actin mRNA as a control for lane loading. (b and c) Dose–response curves for Cd-induced cell death. Cells were treated for 32 h with the indicated concentration of CdCl2 and cell death monitored by using b, the 3-(4,5- dimethlythiazol-2-yl)-2,5-diphenyl tetrasodium bromide assay, or c, the lactate dehydrogenase release assay. rvZIP8ha cells contain the HA-tagged ZIP8 protein. rvZIP8m cells contain a mutated
ZIP8 cDNA. Data represent means ± SD of triplicate determinations.
Figure 3. Increased Cd uptake caused by membrane-localized ZIP8. (a) Time/dose-dependent
109 CdCl2 uptake into rvZIP8 or rvLUC cells. (b) Western blot of ZIP8ha in microsomes (30 vs. 10
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µg per lane). (c) Western blot of ZIP8ha in cytosol (C) or microsomes (M) (30 µg per lane) from rvLUC or rvZIP8ha cells. Arrow denotes band at 55 kDa. (d) Localization of the ZIP8ha protein. rvZIP8ha cells (Upper) or rvLUC cells (Lower) were fixed and incubated with a primary anti-HA antibody and a secondary goat anti-rabbit FITC-conjugated antibody. Cells were counterstained with propidium iodide (PI) to visualize nuclei. Confocal fluorescent microscopy detected FITC
(Left), PI (Center), or both FITC and PI (Right).
Figure 4. Localization of ZIP8 mRNA from sensitive (D2 and 129S6) versus resistant (B6 and
A/J) inbred mouse strains. (a) Northern analysis of testicular ZIP8 mRNA by using poly(A+) RNA.
ACTB, -actin mRNA as a control for lane loading. (b) In situ hybridization of ZIP8 mRNA in testis of the four inbred strains. Left show hematoxylin-and-eosin tissue staining (bright-field),
Center show signal from in situ hybridization (dark-field), and Right show both images overlaid.
Arrows show ZIP8 mRNA localized in the vascular endothelial cells of D2 and 129S6, a feature not detected in B6 or A/J mice. (c) High-magnification bright-field ZIP8 in situ image of testicular capillaries from the indicated mouse strains. (Lower) Photographic emulsion showing capillary in situ pattern (Left) was stripped and section-stained with an anti-CD31 endothelial-specific antibody that was detected by using a biotinylated secondary antibody and Alexa Fluor-488 streptavidin.
Antibody reactivity is shown in green. DAPI (blue color) was used as a nuclear counterstain.
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Figure 1.
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Figure 2.
- 34 -
Figure 3.
- 35 -
Figure 4.
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-1 CTGGCTCAGC TAGCCCACCA ACTTGCGACT GATTTGGTTT CCTCTTTCTC 49 CTCTGATTTT TAAGAAGTGA GGCAAAAAAC CAATTCCTGT TCATTTTTGA 99 GACAATGCAA GGGGCTCTAG ACACCGTTGC AGGAAGCTGA TTTCATGGAG Exon 1c 149 GTCACCAAAA AAGTGGGCCG GGACACCAGG AAGGGGGCTA GGGATCCCAC 199 CGTGGAGGTG GGCTCCAGGG AGTAGGCACC ACAGCGGCTG GGATGGCAGA 249 CAGAGAGGTA AGCGCCCCGG CGTGGACGCA CTTGGGAGAT CAGCCTGGGG 299 GAGTGTGACA CCACGTGCTT CAGGGGCGGG GACAACACTC TCCCTCCCCT 349 TCTTATCTCT AAAGGGTGCA GGGCCTGATC TCAGAGTTTC TTAGAGAAAG 399 GAGAGAGAAA GACAGACAGA GAGAGAGAGA GAGAGAGAGA GAGAGAGAGA Exon 1b 449 GAGAGAGAGA GACATAGACA GTAGAGGTGA GAGGCTAGCA ACTTCTCCGT 499 TTCAACTTGC CAAGCTCACA GCCAGTTGTG CAGAGAGGGT CCAGCGTAGA 549 CGCGGGAACC TGGGGACCCC GAGGGAGTGT TTCTCTGCAG ATTTTTGGGG 599 GGTGGGAGCT TCAGTCCCGA CCCTTCCACG TTTCTGCGGC GGGAAAAAAA 649 AGCCTCAAGA CAGGAAGAGG AGTTGGGGTT GCACAGTCAG AGGATTTAGT 699 TCCCCGAAGT GTGGCGCGGT TGGGGCTTTA TATCCGGGAC AAGCGAGAGT Exon 1a 749 CACCACACTG TAAGCAAATC CTGGGGCTTC AGCAAACCAG GCTCCGGGCA 799 GCTGGCCCCA CGCCCGGCTC GGAGTTCGAG ACTCTGGCCA CTCAGAGCTC 849 TGGGGCCCGC AGACTGCTGA CCCAGTGCCC TTTATCTCTG TCCCCCTTTG 899 TTCCTTTTAT CTCAGGCTCC GCAGGAGGCT CCGAGAGGCC ACACTGCCTA 949 TCGCCCTTCT GGCTGTTCTA CCTCTTCAAT GCCCCGAAGG TCTGAGACTG 999 CAGAGCGGGG GCACGGAGTG TCTCGCTAAC TGGGCCAGGC AGTGACGCGG Exon 2 1049 GACCGGCGCA CCCTCCCACC CCCACCCTAA CGGCACTGAC ACCGGGGCAC 1099 CGGCGCGGGT CCCTGCATCA GCTAGCTCCG GAGCTGCGCG CTCCACGATG
Figure 5. DNA sequence upstream of exon 2 of the mouse Slc39a8 gene, showing the three alternative first exons (bold). Translation of the ZRT-, IRT-like protein (ZIP)8 protein begins at
+233 of exon 2; the initiation codon ATG is underlined. Note that all three introns after the alternative exons 1 have acceptor sites GT that can join with the donor site AG immediately before exon 2. The remaining portions of exon 2 and exons 3–9 are not shown. The human SLC39A8 gene likewise has three exons 1 (data not shown).
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Table 1. Primers used in progeny from the B6D2F1 x B6 backcross
Markers Primer-F (sequence) Primer-R (sequence) D3Mit110 AGCCCTAGGATTTGCATGG TTGGGGTTTTGACTTTTATTTATT M15-2 AGGTGCCAGAGCCAAAAA GACAGGGGAAACAGAACTATCC M13-2 TCATGTTGTATTTATGCACTTAGGA ACTTTGGACATAGGGAAATTCTG M12-17 GGCTTAAAAGAGAGTACCGGG TTCCACCACTTCCCTTTACAA M12-7 CAAAATGACATAAAACTATCACATGC CATTTCAAAACATCATCCATAAAA M11-22 AAGTGCATCTAAAGAAAGGAAATG CACTTAACAAGTCTTTGAAAATGGA M11-6 TTTATTTCCTTTGAGATGAGGG TGATAAGATGACAATCAATGCTG M49-18 ATTATTGGTGAGGGGCAGAG AATCCATGCATGTGGACGTA S530 AGGGCTGTGCACCTTTCTTA TGGCACTACACTCCCTTTCC S901 CCCTTGCTGATGAAGAGACC CCAGCTCTGCCTAAGCTTGT S101 TCTCCTCTAATGGGCTGGAA TGGTTTACTACCGAGTGAGCA S131 CAGCTCAACAGGAGGGAAAC AAAATCTTGCCACGTGCTCT S132 GTTGGGTGGGAAAACAACAG GGGAACAACCTAAGGGCAAT M49-11 GGGGTTGCATCTGTCTCTTT TCATGCACACACACATGGAC M9-9 TGAATTTCCACTTACAGTTCACTTG TTTCTTCAGTTTCTATAATAACCCCC D3Mit291 AACTTCATGTACTCTCTTTCTCTCAGC CCACAAGTAGGACAACGCAA M10-26 TCTAGAGGAACAGAACAGAAGGA TCAGTTTCTAAGGCAATCTAGTGA D3Mit255 TGACACAAACTTGCATTATCTGG TTACTTAAGAAACTTGCCCTCCC
The positions of these markers can be seen in Fig. 1c.
Table 2. Positions of the informative SNPs when analyzing progeny from the B6D2F1 x B6 backcross
SNP Sequence SNP Sequence S530 GAGCACTGTG(A/G)GGAGGGAGCG S901 GTCACACAAA(A/G)CGAAGGAAAG S101 GAGTACAGCA(A/T)CTGGTGACAT S131 GTCCTCACCC(T/C)TCTGCCAGGA S132 ACCTCAATAC(T/C)GGACACAAGA
The bolded biallelic nucleotide site, e.g. (A/G), in the middle of each primer, denotes the informative SNP that helped in identifying the nine new recombinant offspring
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Table 3. Comparison of the intron-exon structure of the mouse and human SLC39A8 genes
Exon, bp Intron, bp
Number Mouse Human Mouse Human
a a 1c 256 184 bp 708 bp 801
a a 1b 144 139 439 429
a a 1a 37 26 156 159
2 451 696 26,096 28,615
3 163 163 6,812 8,064
4 170 170 1,410 2,326
5 123 123 467 509
6 171 165 25,968 36,239
7 208 208 199 199
8 185 185 2,020 4,298
b b 9 1,587 1,530
c c Total (excluding exons/introns 1) 3,058 3,240 62,972 80,250 aHuman exons and introns 1c, 1b and 1a are theoretical but extracted by human/mouse/rat comparative genomics alignments; the exons and introns match the mouse and rat Slc39a8 sequences sufficiently, but are not yet supported by ESTs in the human database. bLongest possible exon 9 out of many transcripts in the EST database. cLongest possible transcript though numerous shorter ones are found in EST database.
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Chapter III. Introduction to SLC (Solute Carrier) Superfamily and
SLC39 Family Transporters
The Solute-carrier (SLC) Superfamily
With the completion of the human/mouse genome projects, we now have a fairly complete list of genes that can encode proteins. Based on homology analysis and domain structure analysis, genes can be grouped into superfamilies and families. Thus, we can study metal transporting proteins in a thorough and systematic way.
Proteins with transport functions can be roughly classified into 3 categories: ATP-powered pumps, ion channels, and transporters. Pumps utilize the energy released by ATP hydrolysis to power the movement of the substrates across the membrane, against their electrochemical gradient.
Channels have two states –open and closed. Channels at the open state can transfer the substrates
(ions or water) down their electrochemical gradient, at an extremely high efficiency. Transporters facilitate the movement of a specific substrate –either against or following their concentration gradient; as generally believed, conformational change of the transporter protein is involved in the transfer process.
According to the Human Genome Organization (HUGO) Gene Nomenclature Committee, all human transporters can be grouped into the solute-carrier (SLC) superfamily. Currently, there are
46 families and 360 putatively functional transporter genes in the human genome (Hediger et al.,
2004; http://www.bioparadigms.org/slc/menu.asp). No homology is shared between different SLC families. At least 20-25% amino-acid sequence identity is shared by members belonging to the same
SLC family.
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There are five SLC families that transport Zn and other essential divalent metal cations: the
SLC11 proton-coupled metal ion family (Mackenzie and Hediger, 2004); the SLC30 Zn efflux
(formerly known as the CDF family (Palmiter and Huang, 2004); the SLC31 copper transporter family (Petris, 2004); the SLC39 metal-ion transporter family (Eide, 2004); and the SLC41 MgtE- like magnesium transporter family (Hediger et al., 2004). Outside the SLC superfamily, some pump
ATPases and ATP-binding cassette (ABC) transporters can also transport metals. For example,
Menkes disease and Wilson disease, two human disorders of Cu metabolism, were found to be due to mutational defects in the ATP7A and the ATP7B genes, respectively (Solioz and Vulpe, 1996).
Overview of ZIP (SLC39) Family Transporters
“ZIP” stands for Zrt-, Irt-like proteins, which were the first members of this transporter family to be identified. Zrt1 and Zrt2 are the primary Zn uptake transporters in the yeast Saccharomyces cerevisiae and Irt1 is the major iron uptake transporter in roots of Arabidopsis thaliana. The ZIP family has grown to more than 575 members –including proteins in bacteria, nematodes, insects, and mammals (from Pfam database of Sanger Institute, 05/04/2006: http://www.sanger.ac.uk/cgi- bin/Pfam/getacc?PF02535). Among these, ZupT is a Zn uptake transporter in Escherichia coli
(Grass et al., 2005) and several ZIPs have been implicated in Zn, Fe, and Mn transport in plants
(Eide, 2004). The SLC39 family is therefore evolutionarily an extremely ancient gene family.
Database analyses have indicated that there are 14 ZIP transporter genes encoded by the human or mouse genome, i.e. each of the 14 genes in mouse has an ortholog in human. There have been no
SLC39 genes lost or new ones gained since the human-rodent split ~70 million years ago.
ZIP proteins transport metal-ion substrates across cellular membranes into the cytoplasm.
Whereas many members are involved in the uptake of metal ions across the plasma membrane,
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some are known to efflux metals from intracellular compartments. To date, the biochemical mechanisms involved in this transport have not been extensively investigated.
The ZIP family can be split into several subfamilies, based on a higher degree of sequence conservation within these groups. There are four subfamilies for all the ZIP proteins (Eng et al.,
1998): subfamily I, subfamily II, gufA subfamily, and LIV-1 subfamily. Subfamily I consists largely of fungal and plant members. Subfamily II is a group of mammalian and nematode proteins.
The gufA subfamily is related to the Myxococcus xanthus gufA gene, whose function is still unknown. The founding member of LIV-1 subfamily is the estrogen-regulated gene LIV-1 (ZIP6).
LZT protein is another name for this protein family, which stands for “LIV-1 subfamily of ZIP Zn transporters” (Taylor et al, 2003 review article).
Based on this classification system, the fourteen human/mouse ZIP proteins are classified into either Subfamily II or the LIV-1 subfamily. For example, ZIP1, ZIP2, ZIP3, ZIP9, and ZIP11 fall in subfamily II. ZIP1, ZIP2 and ZIP3 are further grouped together in that they all share the conserved
12-amino acid signature sequence, HSVFEGLAVGLQ, within the fourth transmembrane (TM) domain (Dufner-Beattie et al., 2003a). ZIP4, ZIP5 (Dufner-Beattie et al., 2004; Wang et al., 2004a;
Wang et al., 2004b), ZIP7 (Taylor et al., 2004), ZIP8 (Taylor et al., 2003), ZIP14 (Taylor et al.,
2005), and the predicted genes ZIP10, ZIP12, and ZIP13 (OMIM database) (Taylor and Nicholson,
2003) are all included in the LIV-1 subfamily. The subfamily members contain a signature motif
(H/EEXPHEXGD) in TM domain V (Taylor et al., 2005).
Most ZIP proteins have eight TM domains and similar predicted topologies, with their N- terminus and C-terminus outside the cells. Some ZIP proteins (e.g., ZIP4) have long N-terminal domains. Whereas most loops between TM domains are quite short, a longer loop region is frequently found between TM domain III and IV. This region often contains a histidine-rich region
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with the sequence (HX)n where n generally ranges from 3 to 5. The function of this region is not yet clear. Determinants of substrate specificity have been mapped to the extracellular loop between TM
II and III in the Irt1 protein (Rogers et al., 2000). For the ZIP6 protein, this region is also rich in histidine residues. TM domains IV and V are particularly amphipathic and contain conserved histidine residues, frequently with adjacent polar or charged amino acids. Given their sequence conservation and amphipathic nature, TM IV and V are predicted to line a cavity in the transporter through which the substrate passes. Consistent with this hypothesis, conserved residues in these regions are essential for function (Rogers et al., 2000). It is also interesting to note that the greatest degree of conservation among ZIP proteins is found in the TM domains (Eng et al., 1998).
Introduction to Individual Human/Mouse ZIP proteins
The ZIP1, ZIP2 and ZIP3 proteins are all grouped in ZIP protein subfamily II, and all contain the conserved 12-amino acid signature sequence, HSVFEGLAVGLQ, within TM domain IV
(Dufner-Beattie et al., 2003a). ZIP1 mRNA is abundant in many mouse tissues, whereas ZIP2 and
ZIP3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted. The abundance of ZIP1, ZIP2 and ZIP3 mRNAs in the intestine and visceral endoderm are not regulated by dietary
Zn (Dufner-Beattie et al., 2003a); this is in direct contrast to ZIP4. These studies suggest that ZIP1,
ZIP2 and ZIP3 transporters may play cell-specific roles in Zn homeostasis rather than primary roles in the acquisition of dietary Zn –as ZIP4 does.
PC-3 cells transfected with the human ZIP1 cDNA and protein exhibited increased uptake of Zn.
The Vmax was increased with no change in Km (Franklin et al., 2003). K562 cells transfected with
65 65 human ZIP2 accumulated more Zn than control cells; the apparent Km was 3 mM. This Zn
- uptake could be inhibited by several other transition metals, and stimulated by HCO3 treatment
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(Gaither and Eide, 2000). A vesicular localization of ZIP1, corresponding partially to the endoplasmic reticulum, was observed in the transfected COS-7 cells (SV40 transformed African green monkey kidney fibroblast cells), PC-3 cells (a prostate cancer cell line), and several other epithelial cell lines (Milon et al., 2001). However, in transiently transfected polarized human intestinal Caco-2 cells, the Myc-tagged human ZIP1 proteins were expressed at the apical membrane (Cragg et al., 2002). And in the non-adherent K562 erythroleukemia cell line, the expressed recombinant human ZIP1 protein was found to be mainly localized to the plasma membrane (Gaither and Eide, 2001).
The peripheral zone is the major region of the human prostate gland where malignancy develops.
The normal peripheral zone glandular epithelium has the unique function of accumulating high levels of Zn. In contrast, the ability to accumulate Zn is lost in the malignant tumor cells. There is a report suggesting that the down-regulation of ZIP1 in adenocarcinomatous glands is an early event in the development of prostate cancer (Franklin et al., 2005). In normal peripheral zone glandular epithelium and in benign hyperplastic glands (also Zn-accumulating), ZIP1 and cellular Zn were prominent. Both were markedly down-regulated in adenocarcinomatous glands and in prostate intra-epithelial neoplastic (PIN) foci. These changes occur early in malignancy and are sustained during its progression in the peripheral zone. Interestingly, ZIP1 was found to be expressed in the malignant cell lines LNCaP, PC-3, DU-145 (Zn-accumulating human prostate cell lines), as well as in the nonmalignant cell lines (Costello et al., 1999; Franklin et al., 2005). There is also evidence of
ZIP2 expression in LNCaP and PC-3 cell lines (Costello et al., 1999). But another study showed that only ZIP1, and no ZIP2, is expressed in the LNCaP and PC-3 cell lines (Franklin et al., 2003).
Zn has previously been demonstrated to be a potent inhibitor of osteoclastogenesis and osteoclast function. Osteoclasts were shown to express ZIP1, which was “diffusely” distributed
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throughout the cytoplasm. Following an adenoviral-mediated overexpression of ZIP1 in murine osteoclasts, ZIP1 was predominantly co-localized with actin at the sealing zone, where it significantly inhibited osteoclast function (Khadeer et al., 2005). This overexpression negatively impacted NF-kB binding activity. The other side of the story is that Zn deficiency is associated with retardation of bone growth. Two Zn transporters were identified in both human mesenchymal stem cells and in osteoblastic cells—ZIP1 and ZIP6 (LIV-1). During the differentiation process of pluripotent mesenchymal stem cells into osteoblast-like cells, both Zn uptake and expression of the
ZIP1 protein were increased. Overexpression of ZIP1 in mesenchymal stem cells resulted in
Alizarin red-positive mineralization and increased expression of specific osteoblast-associated markers (Tang et al., 2006). An siRNA-mediated reduction of ZIP1 protein expression in mesenchymal stem cells caused decreased Zn uptake and inhibition of osteoblastic differentiation under osteogenic culture conditions.
During lactation, a substantial amount of Zn is secreted into milk. Thus, the mammary gland has a unique requirement for Zn. ZIP3 was shown to be expressed in the mammary gland; there is also a change in its expression level and subcellular localization during lactation (Kelleher and
Lonnerdal, 2003). With HC11, a unique mammary epithelial cell model that can respond to prolactin, ZIP3 was demonstrated to play a major role in Zn uptake into mammary epithelial cells
(Kelleher and Lonnerdal, 2005).
ZIP3-null mice are viable and fertile. Under normal growth conditions, they exhibit no obvious phenotypic abnormalities. Deletion of ZIP3 does not alter Zn homeostasis. In knockout mice, stressed with a Zn-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
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noted. These results suggest that this protein plays an ancillary role in Zn homeostasis in mice
(Dufner-Beattie et al., 2005).
ZIP4 plays an essential role in dietary Zn uptake. This role is illustrated by the human disease acrodermatitis enteropathica, due to Zn deficiency (AEZ). AEZ is caused by the reduced uptake of dietary Zn by enterocytes, and the ensuing systemic Zn deficiency leads to dermatological lesions and immune and reproductive dysfunction. The gene responsible for AEZ was identified to be human ZIP4 (Kury et al., 2002). Consistently, expression of the mouse ZIP4 is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by Zn. Dietary Zn deficiency causes a marked increase in the accumulation of ZIP4 mRNA and protein in these tissues and, more specifically, on the apical side of the epithelial cells in these tissues (Dufner-Beattie et al., 2003b).
ZIP4 recombinant protein could lead to increased Zn uptake in transfected cells (Kim et al.,
2004; Wang et al., 2004a). The expressed recombinant protein was largely present in intracellular organelles in transfected cells grown in Zn-replete medium. Upon Zn deprivation, ZIP4 rapidly transit to the plasma membrane. Increased surface levels correlated with increased Zn uptake activity. Conversely, treating cells with low micromolar Zn concentrations stimulated the rapid endocytosis of this transporter (Kim et al., 2004). Zn-responsive protein trafficking might be a conserved mechanism controlling activity of many mammalian Zn uptake transporters. In addition to ZIP4, ZIP1 and ZIP3 were also shown to be responsive to Zn (Kim et al., 2004; Wang et al.,
2004a).
Six AEZ-associated human missense mutations were introduced into the orthologous mouse
Zip4 gene and expressed in cultured cells (Wang et al, 2004). All mutations decreased 65Zn uptake activity. The mutants fell into two groups. Several alleles (G340D, L382P, G384R, and G643R)
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failed to localize on the cell surface at high levels. These defects were attributed to defective folding and/or improper localization in the secretory pathway. Two other alleles (P200L and G539R) led to only 70% of Vmax and unaltered apparent Km of the wild-type protein. Both mutant proteins accumulated to normal high levels in the plasma membrane, but showed a slower endocytosis rate, which is independent of Zn status.
The mouse ZIP5 transporter prefers Zn over many other potential metal ion substrates (Wang et al., 2004b). ZIP5 shares homology with the ZIP4 protein, but, in great contrast to ZIP4, ZIP5 localizes specifically to the basolateral membrane of MDCK cells in cell cultures (Wang et al.,
2004b); immunohistochemistry also localized ZIP5 to the basolateral surfaces of enterocytes, acinar cells, and visceral endoderm cells in mice fed a Zn adequate diet. Whereas ZIP4 is induced and recruited to the apical surface of enterocytes and endoderm cells, ZIP5 is removed from these cell surfaces and internalized during dietary Zn deficiency (Dufner-Beattie et al., 2004). In the pancreas, ZIP4 is expressed in beta-cells, whereas ZIP5 is expressed in acinar cells. The high level of ZIP5 expression in the pancreas may provide serum Zn to these tissues for later excretion into the gut. Thus, ZIP5 might be a central player in mammalian Zn metabolism.
The SLC39A6 gene, encoding ZIP6 (LIV-1), has been previously associated with estrogen- positive breast cancer and its metastatic spread to the regional lymph nodes. Expression of ZIP6 in
CHO cells confirmed its role as a Zn-influx transporter. Recombinant ZIP6 locates to the plasma membrane, and concentrates in lamellipodiae. It is expressed in a number of different tissues with highest levels in the breast, prostate, placenta, kidney, pituitary and corpus callosum, and lowest levels in the intestine and heart, as well as widespread low expression in the brain. The ZIP6 protein sequence contains a signature motif (HEXPHEXGD) in TM domain V (both histidine residues were speculated to be essential for Zn transport; later on, it was demonstrated that only the second residue
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was essential). This motif has identified a new subfamily of ZIP transporters, which was termed
LZT (LIV-1 subfamily of ZIP Zn transporters) (Taylor and Nicholson, 2003).
The ZIP7 (KE4) protein is also classified in the LZT protein family (Taylor et al., 2004).
Transiently expressed V5-ZIP7 (human) fusion protein in the CHO cells was found to be located on intracellular membranes, possibly including the ER, Golgi apparatus, etc (Taylor et al., 2004;
Huang et al., 2005). Consistent with this observation, endoglycosidase PNGase F did not produce any reduction in the molecular weight of the ZIP7 band in Western analysis. However, even with this intracellular localization pattern, over-expression of ZIP7 can still increase the intracellular free
Zn, as measured by use of the Zn-specific fluorescent dye Newport and zinquin staining (Taylor et al., 2004; Huang et al., 2005). These results indicate that ZIP7 is capable of transporting Zn from the Golgi apparatus into the cytoplasm of the cell. By being expressed in the yeast mutant strain zrt3
(which is defective in the release of stored Zn from vacuoles), ZIP7 was found to be able to decrease Zn accumulation in the Golgi and increase the nuclear/cytoplasmic labile Zn level (Huang et al., 2005). In cultured cells, Zn-rich condition inhibits the protein expression of endogenous ZIP7, but has no effect on the gene expression and intracellular localization. ZIP7 is ubiquitously expressed in human and mouse tissues –with heart, skeletal muscle, kidney and liver as the organs having the highest levels of expression (Huang et al., 2005).
A human ZIP8 (BIGM103) transcript with three-protein coding potential was identified (Begum et al., 2002), and the expressed proteins from the longest ORF showed similarities with ZIP family proteins. In ZIP8-overexpressed CHO cells, intracellular Zn accumulation was found to increase.
The expressed protein possesses the hallmark of Zn-metalloproteinase, which classifies it into the
LZT subfamily (Taylor and Nicholson, 2003; Begum et al., 2004). Expression of this transcript is very low or undetectable in unstimulated monocytes, whereas a steady expression level was
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observed under a variety of conditions: during differentiation of monocytes to dendritic cells and macrophages, with the stimulation of the mycobacterium BCG cell wall, with bacterial cell wall stimulation, and during exposure to inflammatory cytokines such as TNFa.
ZIP14 has the signature metalloproteinease motif HEXPHEXGD, but does not have the initial histidine residue. However, ZIP14 is still able to transport Zn in a temperature-dependent manner. It is localized to the plasma membrane and glycosylated (Begum et al., 2004; Taylor et al., 2005).
ZIP14 may play a role as a Zn transporter during the early stages of adipogenesis (Tominaga et al.,
2005); it is quickly elevated by the addition of adipogenesis inducers to mouse 3T3-L1 preadipocyte cells. ZIP14 was highly restricted to the potential differentiation state of 3T3-L1 cells, and the expression level was quite low in the nonadipogenic NIH-3T3 cells.
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Chapter IV. ZIP8, member of the solute-carrier-39 (SLC39) metal
transporter family: characterization of transporter properties
Abstract Cadmium (Cd2+, Cd) is a dangerous metal distributed widely in the environment. Recently, this laboratory identified the ZIP8 transporter protein, encoded by the mouse Slc39a8 gene, to be responsible for genetic differences in response to Cd damage of the testis. Stable retroviral infection of the ZIP8 cDNA, in mouse fetal fibroblast (MFF) cultures (rvZIP8 cells), leads to as much as a
10-fold increase in the rate of intracellular Cd influx and accumulation. In the present study we show that Cd uptake operates maximally at pH 7.5 and temperature of 37 ºC, and is inhibited by cyanide. Of more than a dozen cations tested, manganese( Mn2+, Mn) is the best competitive cation
+2 +2 for Cd uptake. The Km for Cd uptake is 0.62 mM and the Km for Mn uptake is 2.2 mM; thus, Mn is likely the physiological substrate for ZIP8. Cd uptake is independent of sodium, potassium or chloride ions, but strongly dependent on the presence of bicarbonate. By Western blot analysis of rvZIP8 cells, we show that ZIP8 protein is glycosylated. Using Z-stack confocal microscopy in
Madin-Darby canine kidney (MDCK) polarized epithelial cells, we found that ZIP8 is localized on the apical side––implicating an important role for Mn or Cd uptake and disposition. It is likely that
2+ - - ZIP8 is a Mn /HCO3 symporter, that a HCO3 gradient across the plasma membrane acts as the driving force for Mn uptake, and that Cd is a rogue hitchhiker displacing Mn to cause Cd-associated disease.
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(Introduction)
Cd is a highly toxic metal––widely distributed in contaminated soil, cigarette smoke, toxic waste dump sites, and polluted sea food. Acute exposure to large Cd doses can result in damage to the central nervous system, gastrointestinal tract, lung, liver, bone, ovary, placenta, and testes
(Waisberg et al., 2003; Zalups and Ahmad, 2003).
Chronic exposure to low doses of Cd results predominantly in nephropathy and osteomalacia;
Cd is eliminated slowly and thus accumulates with age. The level of Cd in the environment has risen with the rise of industrialization, and Cd-induced human disease is of growing concern. The increasing levels of environmental Cd, in combination with longer life expectancy, work together to enhance the body’s Cd burden: the average accumulation of Cd in the kidneys of a person who smokes at least two packs a day for 50 years, for example, is beyond the threshold sufficient for causing overt Cd nephrotoxicity (http://www.trace-elements.org.uk /cadmium.htm).
Cd is classified as an IARC Category I human carcinogen. People who are at highest risk for
Cd-associated lung cancer include cigarette smokers, women with low body-iron stores, people with a habitual diet rich in high-fiber foods and contaminated shellfish, and malnourished populations
[reviewed in (Jarup et al., 1998; Waalkes, 2003).
Because Cd is not essential to living organisms, Cd-transporting proteins are expected to be transporters of one or more essential metal ions. There have been numerous studies about Cd transport in bacteria, yeast and plants; until recently, however, very little had been known in vertebrates about the molecular mechanisms of Cd transport. Focusing on testicular necrosis as a sensitive endpoint for genetic differences in response to Cd, this laboratory set out to identify the
Cdm locus––defined more than 3 decades ago (Taylor et al., 1973) . Using mouse genetics, recombinant inbred lines and advances in knowledge about the mouse genome, this laboratory
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demonstrated that the Slc39a8 gene is the Cdm locus responsible for genetic differences in damage to the vasculature endothelium specific to the testis (Dalton et al., 2000b; Dalton et al., 2005).
Slc39a8 is one of 14 members of the mouse Slc39 gene family. The human SLC39 gene family also has 14 members, all of which are orthologous and highly conserved with the mouse genes. The
Slc39 genes encode Zrt-like, Irt-like proteins (ZIP), first characterized in Saccharomyces cerevisiae and Arabidopsis thaliana; the gene product of the mouse Slc39a8 gene is thus called ZIP8 (Eide,
2004). This laboratory has produced a stable retrovirally infected ZIP8 cDNA into mouse fetal fibroblast (MFF) cultures, to generate ZIP8-expressing rvZIP8 cells, which exhibit as much as a
10-fold increase in the rate of intracellular Cd uptake and accumulation and a ~30-fold increase in sensitivity to Cd-induced cell death (Dalton et al., 2005).
In the present study, we have characterized further the membrane-bound ZIP8 transporter protein. These data include: dependence of Cd uptake by ZIP8 on temperature, cellular ATP-
- mediated energy, pH, and HCO3 ; cation competition studies; effects of cation or anion substitution or depletion; glycosylation; and membrane localization of the transporter.
Materials and Methods
Chemicals. CdCl2, MnCl2 and ZnCl2 were bought from Fisher Scientific (Pittsburgh, PA).
Bovine serum albumin and the remainder of the chemicals––including Chelex 100––were
109 purchased from Sigma (St. Louis, MO). Cd uptake studies were performed with CdCl2 [710 mCi/mg (1 mCi = 37 mBq) in 0.1 M HCl], purchased from Amersham Biosciences (now GE
54 Healthcare; Piscataway, NJ). Mn uptake studies were carried out with MnCl2 (7,734 mCi/mg in
0.5 M HCl), purchased from PerkinElmer (Wellesley, MA). Zn uptake studies were done with
65 ZnCl2 (140 mCi/mg in 0.1 M HCl), purchased from the National Laboratory of Oak Ridge (Oak
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Ridge, TN). Transporting medium for these divalent metal ions was a modified version of Hank’s balanced salt solution (HBSS), shown in Table 1.
Cell Culture and Transfection Methods. MFF cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) (Invitrogen; Carlsbad, CA) plus 10% fetal bovine serum (FBS) from
Hyclone (Logan, UT) at 37°C in 5% CO2. All culture medium contained 100 Units/ml of penicillin and 100 µg/ml streptomycin. The ZIP8-infected cells were maintained in selection medium (Dalton et al., 2005) supplemented with hygromycin (400 µg/ml) and puromycin (3 µg/ml). Transfection was carried out, following the manufacturer’s protocol for Lipofectamine 2000 (Invitrogen).
Cloning of the ZIP8 cDNA and Delivery into MFF Tet-Off Cells. Oligo-dT-primed reverse transcription was carried out with C57BL/6J mouse testis total RNA. Primers for amplification began at the start codon and ended at the stop codon; a consensus Kozak sequence at the start-site was included for efficient expression. For ZIP8, restriction sites were added at the 5' (Bam HI) and
3' (Cla I) ends of the coding sequence for cloning into the pRevTRE vector (Invitrogen); for ZIP4, the sites are 5' (Bam HI) and 3' (Sal I). In each 3' primer, the coding sequence of a hemagglutinin
(ha) tag was also inserted in-frame, before the termination codon of that protein coding sequence. A
MFF cell line was generated by continuous passage of primary MFF cultures. These cells were infected with a retrovirus encoding the Tet-off receptor (Bergwitz et al., 2000) and a clone was selected using puromycin (3 µg/ml) resistance. These cells were infected with retrovirus (rv)- encoding control luciferase (LUC), ZIP8ha, or ZIP4ha cDNAs, to generate respectively the stable rvLUC, rvZIP8, and rvZIP4 cell pools, which were selected for hygromycin (400 mg/ml) resistance
(Dalton et al., 2005). These cell pools constitutively express the transporters via retroviruses expressed from pRevTRE.
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Determination of Cd, Zn and Mn Uptake. The cells were seeded at a density of ~80% confluency in 24-well plates. The next day, the DMEM was replaced with modified HBSS as the
109 54 65 transport medium, unless otherwise specified; CdCl2, MnCl2 or ZnCl2 was added to the transport medium to make the final concentrations 0.25 mM, 0.25 mM or 10 mM, respectively, so that each well contained 0.5 ml transport medium and 0.1 mCi/ml radioactivity. The cells were then incubated for 20 min at 37°C. For each sampling point, the uptake was stopped by placing the plate on ice and quickly removing the transport medium. The cells were washed three times with 0.5 ml cold phosphate-buffered saline (PBS) (containing 1 mM EDTA). After the final wash, 0.5 ml of 0.5
N NaOH was added into each well, and the plates were then incubated at 37°C overnight to digest the cells. The next day, 200 ml of cell lysate from each well was used for liquid scintillation counting, whereas the remaining portion of each sample was used for determination of protein concentration, measured by the BCA protein assay.
Measurement of Metal-Induced Cell Death. The viability of cells was assessed, following exposure to metal ions including Cd2+, Co2+, Cs2+, Cu2+, Fe3+, Fe2+, Hg2+, Mn2+, Ni2+ and Zn2+. All cations were in the chloride form. Pb2+ and Ag1+ could not be precisely tested because of precipitation with chloride ions present. Cell viability was determined as the cleavage of 3-(4,5- dimethlythiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT), as described by the manufacturer’s protocol (Promega; Madison, WI). The Hoeschst assay replaced the MTT assay in studying Cu2+ toxicity, because Cu2+ interferes with the MTT assay. Briefly, after incubating the cells with Cu2+ for 32 h, the cells were washed once with PBS; the cells were fixed with 75% ethanol at –20°C for
1 h. Next, the ethanol was removed, and the wells were air-dried in the hood. Then 250 ml of
Hoechst 33258 solution (5 mg/ml) was added, and the cells were incubated at room temperature in
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the dark for 20 min. Fluorescence was determined in a Wallac Victor2 1420 multilabel counter
(PerkinElmer) at 355 nm excitation and 460 nm emission.
Western Immunoblot Analysis of ZIP8 Protein Glycosylation. Cell pellets were dissolved in denaturing buffer (0.5% SDS and 1% b-mercaptoethanol), and protein concentrations of each cell lysate sample were determined. Cell lysate (5 mg protein) was denatured at 37°C for 30 min.
Nonidet P-40 and sodium phosphate (pH 7.5, 25°C) were added to make final concentrations of 1% and 50 mM, respectively. PNGase F (500 U, New England Biolabs; Ipswich, MA) was added, and the mixture was incubated at 37°C for 2 h. Samples were then denatured with SDS-PAGE loading buffer (37°C for 30 min), and then run on a 10% SDS-PAGE gel before immunoblotting. Next, these protein samples, separated by SDS/PAGE gels, were transferred to nitrocellulose and blotted
(Dalton et al., 2005). For detection, a rabbit affinity-purified polyvalent anti-ha antibody (a-ha;
Bethyl Laboratories; Montgomery, TX) was used at 1/5,000 dilution.
Z-Stack Confocal Microscopy for Detecting Cell Surface-Bound ZIP Proteins. Zn depletion of FBS was carried out, using Chelex 100. Briefly, a solution of 5% (w/v) Chelex 100 resin in FBS was incubated overnight, with constant stirring, followed by filtration through a 0.2-µm filter. This
FBS was then added into DMEM to make Chelex 100 medium. Madin-Darby canine kidney
(MDCK) cells (ATCC; Manassas, VA) were seeded onto (non-coated) cover slips in a 24-well plate.
The next day, each well of cells was transfected with the combination of the following plasmids–– pRevTet-Off and pRevTRE-ZIP8––according to the manufacturer’s transfection protocol for
Lipofectamine 2000 (Invitrogen). Some wells of these cells were transfected with the plasmid
+ - encoding green fluorescent protein fused with Na -HCO3 cotransporter-1 protein GFP-NBC1 (Li et al., 2004). Two days post-transfection, the cells were incubated in Chelex 100 medium for 1 hour, and then fixed with 3% formaldehyde for 20 min. Next, the cells were permeabilized with 0.1%
- 55 -
Triton X-100 for 4 min and blocked with 10% FBS-containing PBS medium for 1 hr. Fixation, permeabilization, and blocking were all done at room temperature. The a-ha (Bethyl Laboratories), at 1:500 dilution, was incubated at 4ºC overnight with cells in 1% bovine serum albumin containing
PBS. The next day, the primary antibody solution was removed, and the cells were washed with
PBS three times, 5 min each time. The secondary antibody Alex488-a-rabbit or Alex568-a-rabbit
(Molecular Probes; Eugene, OR) was incubated with the cells at room temperature for 1 h. Next, cells transfected with GFP-NBC1 were mounted for confocal analysis; cells which were not transfected with GFP-NBC1 were co-stained with an apical membrane marker peanut agglutinin
(PNA)-lectin (Molecular Probes) at 1/200 dilution for 1 h at room temperature, and then mounted for confocal analysis. Images were taken on a Zeiss LSM510 confocal microscope (Carl Zeiss;
Oberkochen, Germany). Both Z-line and Z-stack images were obtained, using the LSM5 Image software to analyze specific cellular membrane targeting.
Statistical Analysis. Statistical significance between groups was determined by analysis of variance between each group and Student’s t-test. All assays were performed in duplicate or triplicate, and repeated at least twice. Statistical analyses were performed with the use of SASâ statistical software (SAS Institute Inc.; Cary, NC). The determinations of Km and Vmax values for
ZIP8, and TD50 values for divalent cation-induced cell death, were determined using Sigma Plot
(developed by Jandel Scientific; purchased by SPSS Inc., Chicago, IL; and sold by Systat Software,
Inc., Point Richmond, CA).
Experimental Results:
We have previously shown that ZIP8 is a robust rogue Cd transporter and is not expressed in
MFF cells, addition of a C-terminal ha tag does not alter Cd transport by ZIP8, and rvZIP8 cells
- 56 -
possess Cd uptake properties that are clearly separable from wild-type Tet-off cells or the control rvLUC cells (Dalton et al., 2005). Therefore, rvZIP8 cells or rvZIP8ha cells may be used interchangeably to investigate the transport properties of ZIP8.
Temperature-Dependent Transport. Temperature sensitivity is a salient feature of many carrier-mediated, energy-dependent transport processes. ZIP8-mediated Cd uptake was more than four times greater at 37ºC than at 25ºC (Fig. 1A). At 4ºC, ZIP8-mediated Cd transport was indistinguishable from that seen in rvLUC cells, and very close to background. Between 25ºC and
37ºC, we found that the temperature coefficient Q10 (reviewed in (Bennett, 1984)); http://www.csupomona.edu/~seskandari/Q10.html) was 3.25 for rvZIP8 cells. A Q10 of ~1.0 represents the physical diffusion of ions or molecules, whereas a Q10 of >2.1 denotes an energy- dependent activity. These data confirm that the ZIP8 transporter-mediated process requires cellular energy. Cd uptake by rvLUC cells was only slightly influenced by temperature changes.
ATP-Dependent Transport. An active transporter can move its substrate “uphill,” or against, a concentration gradient of the substrate. The driving force can be the energy from ATP hydrolysis
(in this case, the transporter being called a “pump”); the driving force can also be the energy stored in the electrochemical gradient of a coupled substrate. In this case, a “symporter,” carries two substrates in the same direction, whereas an “antiporter” carries two substrates in opposite directions. Symporters and antiporters are both examples of cotransporters.
KCN is a strong inhibitor of the mitochondrial respiratory chain and significantly decreases
ATP production––thus further disrupting the electrochemical gradient across the cell membrane.
109 KCN (0.5 mM) was added to rvZIP8 cells at 5, 15 or 30 min prior to the addition of CdCl2 (Fig.
1B); at 5 min of pretreatment, KCN completely inhibited Cd uptake. KCN had no effect on Cd uptake in the control rvLUC cells, even during the longest incubation period of time. These data,
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combined with the Q10 temperature coefficient data, confirm that Cd uptake is an active process, requiring energy––either directly from ATP, or indirectly from energy stored in the electrochemical gradient across the plasma membrane.
Dependence of Transport on pHout (pH of the transporting medium). Some cotransporters use protons as the coupled ion. The concentration gradient of protons across the cell membrane provides energy for the transport of the substrate. The SLC11A2 (DMT1) protein, for example, cotransports divalent metal ion (such as Fe2+ or Cd2+) with protons into the cells; as would be expected, SLC11A2 functions optimally at pHout 5.5 (Mackenzie and Hediger, 2004). We adjusted the pH of the uptake medium at intervals between 5.5 and 8.0 and examined Cd uptake (Fig. 1C).
Following incubation, the pH of uptake solutions was again measured and, under these conditions, did not vary by more than 0.1 pH units. Control rvLUC cells, as well as rvZIP4 cells, had only a slight change in Cd uptake. On the contrary, rvZIP8 cells were very sensitive to pH changes, having a maximal activity at pH 7.5––which is the physiological pH of most tissue fluids. This result indicates that, unlike SLC11A2, the ZIP8 protein is likely not to be a proton-coupled transporter.
Metal Cation Competition Experiments. Fig. 2 shows the degree to which each of ten metal ions competes with Cd uptake. We found that Mn2+and Hg2+ were the two best inhibitors, with the order of the inhibitory effect as: Mn2+ > Hg2+ >> Pb2+ = Cu2+ = Zn2+ = Cs2+. This result suggests that ZIP8 may have a very high affinity for Mn2+.
Cd and Mn Uptake Kinetics. Cd was shown previously to be taken up by rvZIP8 cells (Dalton et al., 2005). When the rvZIP8 cells were studied in HBSS, the kinetics of Cd transport (Fig. 3A) confirmed that the process fits the Michaelis-Menten model very well, with a Hill coefficient of
0.92, a Vmax of 92.1 pmol/mg/min, and a Km value of 0.62 mM.
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Because Mn was found to be the most potent inhibitor of Cd uptake (Fig. 2), we carried out direct transport studies with radiolabeled Mn (Fig. 3B). Under our experimental conditions, Mn uptake in rvZIP8 cells was 5-fold greater than that by rvLUC cells, over a broad concentration range.
After subtraction of rvLUC uptake from rvZIP8 uptake, the ZIP8 transporter was found to have a
2+ high affinity for Mn : a Hill coefficient of 0.71, a Vmax of 73.8 pmol/min/mg protein, and a Km of
2.2 mM. These parameters are very similar to that of Cd kinetics, indicating that the ZIP8 protein behaves similarly in transporting either Mn or Cd.
Heavy-Metal-Induced Cell Death. ZIP8, which transports Cd with high affinity, greatly sensitizes cells to Cd-induced cell death (Dalton et al., 2005). We therefore studied rvZIP8 cell viability in the presence of the ten cations that had been tested above for competitive inhibition of
Cd uptake. Figs. 4A, 4B and 4C show that rvZIP8 cells were most sensitive to Cd, and much more
2+ 2+ sensitive than rvLUC cells to Mn or Hg toxicity, with TD50 values of 4 mM vs 11 mM for Mn , and 40 mM vs 65 mM for Hg2+, respectively. No differential toxicity was observed for any of the other metals (data not shown). We found no difference in Zn toxicity between rvZIP8 cells and rvLUC cells (Fig. 4D), even though Zn uptake (at 10 mM) in rvZIP8 cells is at least twice as rapid as that in rvLUC cells (not shown). One possible explanation is that Zn might be complexed or compartmentalized within the cell (Outten and O'Halloran, 2001); thus, an elevation in total cellular
Zn might not mean a proportionate increase in free Zn, and therefore might not reflect a proportionate increase in Zn toxicity. The Fig. 4 data strongly suggest that––in addition to Cd––Mn and mercury are also specific substrates for ZIP8. Furthermore, these results are consistent with the metal ion competition experiments of Fig. 2.
Effects of Sodium, Potassium, or Chloride Substitution on Cd Uptake. ZIP8 has no ATP- binding domains, and no ATP hydrolysis activity has been reported. Therefore, we reasoned that
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ZIP8 is probably a cotransporter that uses an electrochemical gradient of one or more ions as the driving force for the active transport of Cd (and perhaps Mn and other endogenous metals). Sodium
(Na+) is the most commonly coupled ion for animal cells. When Na+ moves into the cell and down its electrochemical gradient, the coupled substrate is “dragged” into the cell, or pumped out of the cells, along with Na+. The greater the electrochemical gradient for Na+, the greater will be the transport rate of the substrate. Therefore, we tested the dependence of Cd uptake on a Na+ gradient by substituting the Na+-containing uptake medium with N-methyl-D-glucamine (NMDG)––an inert organic cation generally believed not to be carried by any transporter (Fig. 5A). We found no change in Cd uptake, whatsoever, indicating that ZIP8 does not cotransport Na+ with Cd.
Animal cells maintain a slight negative charge on the intracellular side of the plasma membrane; this membrane potential is generated by a “leaky” outward potassium (K+) currents, driven by a K+ gradient across the membrane (high K+ inside the cell; low K+ outside). During electrogenic transport processes in which the substrate may be either a charged or neutral molecule, the membrane potential is an important driving force. For example, if the net charge that ZIP8 brings into the cell is positive, an increase of the membrane potential in the negative direction can accelerate the velocity of Cd uptake by ZIP8. Membrane potential can be altered by modifying extracellular K+ concentration. We found that ZIP8-mediated Cd uptake was not influenced by altering the K+ concentration (Fig. 5B); we therefore conclude that ZIP8-mediated transport is independent of K+ and membrane potential alteration.
Chloride (Cl–) ion is another candidate for coupled ion(s). Following the same rationale, we substituted Cl– in the uptake medium with the inert organic anion, gluconate. No influence upon Cd uptake was found (Fig. 5C); thus, we conclude that ZIP8 does not cotransport Cl– with Cd.
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- Dependence of Cd Uptake on Bicarbonate. To test the effects of extracellular HCO3 on Cd
- uptake, we grew the cells in HBSS with differing concentrations of HCO3 , but with the same osmolar pressure and same pH (7.5). Fig. 6A shows that Cd uptake was dependent on the presence
- - of HCO3 ; with increasing concentrations of HCO3 , ZIP8-mediated uptake of Cd increased as well.
- At 4 mM HCO3 , Cd uptake had already reached its maximum. This finding justifies all the earlier
- transporter experiments in this paper, in which 4.17 mM HCO3 in HBSS had been used.
- - The “zero” mM HCO3 in rvZIP8 cells in HBSS (Fig. 6A) is actually not free of HCO3 , because
- - there is exogenous HCO3 from dissolved CO2 in the air, as well as the CO2/HCO3 derived from metabolism in the cells. From the Henderson-Hasselbach equation of this buffer system, pH = pK +
- - log([HCO3 ]/0.03 x pCO2), one can calculate that putatively “HCO3 -free” medium actually contains
- 171 mM HCO3 at 37°C and pH 7.5; this is likely the reason why we found substantial amounts of
- ZIP8-mediated Cd uptake in “~0 mM HCO3 ” HBSS.
- To further confirm the role of HCO3 , we added 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
- (DIDS), a well known competitive HCO3 transporter inhibitor (Cabantchik and Greger, 1992), to
- the 4.17 mM HCO3 -containing HBSS medium prior to adding Cd (Fig. 6B). At 1 mM DIDS, a
- concentration that is regularly used to inhibit 4 mM HCO3 uptake, DIDS was found to impede
- ZIP8-mediated Cd uptake by more than half. When HCO3 concentrations were decreased to negligible amounts (171 mM), low doses of DIDS almost completely abolished ZIP8-mediated Cd uptake: ZIP8-mediated Cd uptake was lowered by 0.25 mM DIDS from 110 to 20 pmol/mg (Fig.
- - 6B). Hence, DIDS is more effective at low HCO3 concentrations than at high HCO3 concentrations.
- These data strongly suggest that ZIP8 mediates HCO3 -dependent Cd uptake, consistent with ZIP8
2+ - being a Cd /HCO3 symporter.
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Glycosylation of the ZIP8 Protein. Many plasma membrane and secretory proteins contain one or more carbohydrate chains linked via asparagine (N-linked) or via threonine or serine (O-linked).
Glycosylation is a common posttranslational modification of proteins in eukaryotic cells. We found two potential N-linked glycosylation sites in the ZIP8 protein sequence, Asn-40 and Asn-88; no predicted O-linked glycosylation sites were found. PNGase F treatment, followed by Western immunoblot analysis, was employed to confirm this prediction. N-glycosidase F, also known as
PNGase F, is an amidase that cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoprotein (Maley et al., 1989). PNGase F will not cleave N-linked glycans containing core a1,3- fucose. Following PNGase F treatment (Fig. 7), there is a significant shift in apparent molecular weight of the ZIP8ha protein band––from ~80 kD to ~50 kD––the latter of which is close to the calculated molecular weight of the core ZIP8ha protein. The ZIP8ha protein is 51.3 kDa with the signal peptide, and 49.5 kDa without the signal peptide. The heterogeneity of ZIP8ha bands in the
80-kD region is likely due to the varying degrees of saturation of available glycosylation sites. It is interesting to note that, following PNGase F treatment of ZIP4ha, on the other hand, any cleavage resulting in the lower-molecular-weight band was negligible.
Localization of ZIP8 to the Apical Surface of Polarized Cells. Western immunoblot analysis had shown only that the ZIP8 protein is detected in the membrane fraction of MFF cells, and confocal images showed clearly that ZIP8 protein is localized, in large part, on the plasma membrane of MFF cells (Li et al., 2004). It should be noted that ZIP8 expression on intracellular membranes was also detected in the cell confocal pictures; this probably represents ZIP8 protein procession and transportation in the endoplasmic reticulum, Golgi apparatus, and “transporting” vesicles.
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Uptake of Cd into living cells is a vectorial process––which happens in the epithelial cells of the small intestine, the lung and the kidney reabsorption tubules. Epithelial cells are polarized cells, with their apical side facing the lumen and the basolateral side facing the basal membrane and tissues; these two sides of epithelial cells have very different expression patterns of transporters, and represent the structural basis for their distinct substrate-handling capacity. In this regard, MFF cells are not the best system to study ZIP8 protein, because they are not polarized cells. We therefore chose Madin-Darby canine kidney (MDCK) cells, a distal convoluted tubule (DCT) polarized epithelial cell line (Simmons et al., 1984), to study further the subcellular localization of the ZIP8 protein.
We confirmed that the ZIP8ha protein is localized predominantly on the MDCK cell surface membrane (Fig. 8). GFP fused to the NBC1 is known to be localized on the basolateral membrane of MDCK cells (Li et al., 2004). Z-stack analysis (Fig. 8, left) clearly shows that the ZIP8 protein localization is distinct from the basolaterally localized GFP-NBC1 protein. Moreover, ZIP8ha was localized to the surface of MDCK cells that is in contact with the culture medium. Taken together, these data suggest that ZIP8 is localized on the apical surface of this polarized epithelial cell line.
This conclusion is further supported by the observation that ZIP8ha is localized to the same cell surface as the apical marker PNA-lectin (Fig 8, right). PNA-lectin is known to bind exclusively to the apical membrane (Cooper, 1984; Li et al., 2004). As recent studies have shown (Li et al., 2004),
PNA-lectin binds to only a subset of MDCK cells and, because transient transfection is only effective in allowing expression in a subset of cells, ZIP8 and PNA-lectin were not easily colocalized. These markers, however, could be visualized in adjacent cells in many microscopic fields, and their localization consistently showed a similar membrane localization pattern.
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In general, the apical-basolateral orientation of a transporter in a polarized epithelial cell of one organ is almost always consistent with the localization pattern in epithelial cells of other organs (Li et al., 2004). Thus, our result implies that the ZIP8 protein takes up endogenous or exogenous metals––from food (in small intestine), blood (in endothelial cells), or glomerular filtrate (in renal proximal or distal tubules)––into tissues or organs where they are distributed.
Discussion
Currently, there are a total of 360 putatively functional genes, divided into 46 families, within the solute carrier gene (SLC) superfamily [http://www.bioparadigms.org/slc/menu.asp]. The ZIP transporter proteins comprise the SLC39 family, having 14 members––highly conserved orthologs–
–between mouse and human. A protein is assigned to a specific family if it shows at least 20-25% amino-acid sequence identity to other members of that family. In plants, several ZIP proteins have been implicated in Zn, Fe2+ and Mn transport (Eide, 2004); some plant ZIP proteins, such as IRT1 and TcZNT1, are capable of transporting Cd (Hall and Williams, 2003). In mammals, all ZIP proteins cloned to date are able to transport Zn at micromolar concentrations; until our recent report
(Dalton et al., 2005), no transport study of Cd by a ZIP protein in vertebrates had previously been reported. ZIP8 is encoded by the mouse Slc39a8 gene.
Our study shows that mouse ZIP8 can transport Cd very efficiently. This uptake process is: energy-dependent; optimal at pH 7.5 (and, thus, ZIP8 is unlikely to be a proton pump); independent
+ + – – of any K , Na or Cl gradient (Fig. 1); and dependent on HCO3 in the transport medium (Fig. 6).
– Physiological concentrations of HCO3 range from ~20 mM in glomerular filtrate, and ~29 mM in extracellular fluids such as blood, to ~12 mM inside most types of animal cells
(http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.table.4057). Thus, it is reasonable to
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2+ - - speculate that ZIP8 is a Cd /HCO3 symporter, with the HCO3 gradient as the driving force for Cd uptake. It has been shown that Zn uptake in yeast by Zrt1 and Zrt2 is energy-dependent (Zhao and
Eide, 1996a; Zhao and Eide, 1996b). Zn uptake by human ZIP1 and ZIP2 has been shown to be independent of K+ or Na+ gradients (Gaither and Eide, 2000; Gaither and Eide, 2001). Furthermore,
- HCO3 dependence has been noted for ZIP2-mediated Zn uptake (Gaither and Eide, 2000).
- In various types of cultured cells, a stimulatory effect of HCO3 on Cd uptake has been reported.
These cells include human erythrocytes (Lou et al., 1991), rat and rabbit erythroid cells (Savigni and Morgan, 1998), and LLC-PK1 cells (epithelial cell line originally derived from porcine kidneys)
- (Endo et al., 1998); these stimulatory effects can be blocked by DIDS, a specific inhibitor of HCO3
-dependent transporters. No specific transporters, however, had been identified in these cell types.
Experiments presented herein may shed light into the underlying molecular mechanism of these
- studies on HCO3 stimulation of Cd uptake.
- The toxicological implications of HCO3 dependence in the intact animal are intriguing. The
- HCO3 concentration is 24 mM in the glomerular filtrate, but is efficiently reabsorbed as it passes through the proximal convoluted tubule (PCT). The concentration drops to ~4 to 6 mM when it reaches the S3 segment, the straight portion of the PCT, and is negligible by the time the filtrate reaches the collecting duct. One major complication of Cd toxicity is kidney damage, which is manifested as proximal renal tubular acidosis. We speculate that the apical localization of ZIP8,
- along with its HCO3 dependence, may explain why Cd damage to the kidney is almost exclusively localized to the PCT: thus, ZIP8 may be the main Cd transporter in the kidney, and it functions well
- only in the PCT due to the presence of high HCO3 concentrations there.
For the first time among mammalian ZIP proteins, our study has demonstrated that ZIP8 has an extremely high affinity for Cd (Fig. 3A). The Km of ZIP8 is only 0.62 mM in our established mouse
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cell culture system, which is half as much as the Km of SLC11A2 (DMT1) for Cd, when the DMT1 transporter mRNA was expressed in Xenopus oocytes (Okubo et al., 2003). SLC11A2 is believed to be important in Cd uptake from the small intestine. Given the fact that SLC11A2 is located apically, it will be important to determine the cell-type-specific expression of ZIP8, because ZIP8 is a likely candidate for Cd uptake from the intestine, lung, glomerular filtrate, and plasma. Indeed, uptake from the plasma into the testis is predominantly mediated by ZIP8 (Dalton et al., 2005). It will also be important to determine the patterns of expression and transport characteristics of the other ZIP transporters.
Cd is not an essential metal. Cd levels in the environment have risen markedly due to industrialization; therefore, Cd has begun to pose a risk to human populations only during the past
150 years. Hence, human and other animals are not expected to be “genetically prepared” to handle this noxious metal, and Cd must be a “hitchhiker” utilizing one or more existing transporters. What, then, are the physiological substrates of mammalian ZIP8?
Our radiolabeled Mn uptake study (Fig. 3B) confirms that ZIP8 has a very high affinity for Mn.
Because Mn is an essential metal, ZIP8 may act principally as a Mn2+ transporter under physiological conditions. The metal concentration and the transporter affinity will, in general,
2+ determine the metal(s) transported. The Km of 2.2 mM for Mn is close to physiological concentrations: Mn in mammalian tissues ranges between 0.3 and 2.9 mg Mn2+/g wet tissue weight
2+ (Rehnberg et al., 1982; Keen and Zidenberg-Cherr, 1994). The Km of 2.2 mM for Mn is also within the same range determined in many cells lines or tissues: 2 mM for HepG2 cells (Finley,
1998); 18 mM for glial cells of the chick cerebral cortex (Wedler et al., 1989); 0.3 mM for rat astrocytes (Aschner et al., 1992); and 32.2 mM for Caco-2 cells (Leblondel and Allain, 1999). Very little is known about Mn transport pathways at the molecular level; Mn competition experiments
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have suggested that some mammalian ZIP proteins might be capable of transporting Mn, but no direct transport study has been reported until the present study. Interestingly, a phenomenon that Cd and Mn might share the same transport mechanism was speculated in studies with a Cd-resistant cell line (Yanagiya et al., 2000)––although their apparent Km values were much lower (40 nM for
Cd2+; 36 nM for Mn2+) than that in the present study (Fig. 3). Rat spinal cord dorsal horn neurons express ancient conserved domain protein-4 (ACDP4); recently, ACDP4 was found by a yeast two- hybrid system to interact specifically with an intracellular metal ion chaperone in human embryo renal cortical HEK293 cells and enhance cell killing by Cu2+, Co2+ and Mn2+ (Guo et al., 2005).
Zn uptake was reported to be increased when human ZIP8, termed BIGM103 in that study, was expressed in Chinese hamster ovary cells (Begum et al., 2002). It is therefore not out of the question that ZIP8 might also be an important Zn transporter in certain cell types devoid of other efficient Zn transporters.
All of the above studies cited are from cell culture experiments. One must be cautious in extrapolating, into the intact animal, the conclusion that ZIP8 is a specific Mn or Zn transporter, because the ability of this protein to transport a particular cation does not necessarily mean that this protein is physiologically well utilized. Maintaining metal homeostasis is essential for the function of many important enzymes, transcription factors, and other subcellular proteins. Disruption of metal homeostasis can sometimes lead to disease, and even death. Thus, Slc39 gene deletion, or overexpression––at the whole-animal level––would be definitive in assessing the physiological role of a transporter. For example, human ZIP4 is a well-established essential Zn transporter, because the loss of ZIP4 function causes Zn-deficient acrodermatitis enteropathica, a disease marked by skin lesions on the extremities, and immune and reproductive dysfunction, due to decreased uptake of dietary Zn by enterocytes in the small intestine (Kury et al., 2002).
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Western blot and confocal analysis of MFF cells have shown that ZIP8 is localized mainly in the plasma membrane (Dalton et al., 2005). In the present study, using MDCK cell cultures as a model system, we have determined that the expressed recombinant ZIP8 protein is localized on the apical side of these polarized epithelial cells (Fig. 8). This observation with ZIP8 is similar to many reports showing that ZIP1, ZIP2, ZIP3, and ZIP4 are located in the apical side of mouse enterocytes and visceral yolk sac epithelial cells. However, ZIP5 is located on the basolateral membrane in enterocytes, acinar cells, visceral endoderm cells, and cultured MDCK cells (Dufner-Beattie et al.,
2004; Wang et al., 2004b). Thus, ZIP5 is the only ZIP protein found so far to be localized on the basolateral side of polarized cells. It is noteworthy that––in addition to the apical localization––
ZIP8 also shows cytoplasmic distribution, raising the possibility of trafficking between the intracellular compartments and the membrane during physiological and pathophysiological states.
The vectorial transport of a substrate, and the apical-basolateral expression pattern of a specific transporter, generally are quite similar in many ways––across various types of epithelial cells. This is one of the reasons why MDCK cells have become the standard model system for studying polarized epithelial cells. The present study supports the notion that most ZIP protein members, including ZIP8, are internalizing metal ions from the environment, from the blood or glomerular filtrate, into tissues such as intestine, lung, pancreas, or kidney tubules. The only exception is ZIP5, which appears to move metal ions in the reverse direction, from these tissues into the blood stream.
Further studies in MDCK cells, Xenopus oocytes, Slc39a8-containing BAC-transgenic mice, and
Slc39a8(-/-) knockout mice are underway to elucidate the physiological importance of this newly characterized ZIP8 transporter protein.
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Acknowledgements:
The preceding chapter was published in Published in Molecular Pharmacology (in press: http://molpharm.aspetjournals.org/cgi/reprint/mol.106.024521v1). The authors are Lei He,
Kuppuswami Girijashanker, Timothy P. Dalton, Jodie Reed, Hong Li, Manoocher Soleimani, and
Daniel W. Nebert. This work was supported in part by NIH Grants R01 ES10416 (D.W.N.), R01
DK62809 (M.S), and P30 ES06096 (T.P.D., D.W.N.)
We thank our colleagues for valuable discussions and critical readings of the manuscript.
Portions of these data were presented at the 44th (March 2005) Annual Meeting of the Society of
Toxicology, New Orleans, LA.
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Figure Legends
Figure 1. Effects of temperature (A), KCN pretreatment (B), and pHout (C) on Cd uptake in cultured rvZIP8 and rvLUC cells. In addition, the rvZIP4 cell line is shown in the pH study. The temperature- and KCN- dependence studies were carried out in DMEM with 10% FBS as uptake medium, to approximate more closely physiological conditions. On the other hand, the pHout study (extracellular pH of the medium) was performed in HBSS, in order to adjust the pH more easily. Asterisks denote significant (P <0.05) differences between rvZIP8 and rvLUC in panels A & B, and between rvZIP8 and both rvLUC and rvZIP4 in panel C
(Student’s t-test).
109 Figure 2. Metal cation competition for Cd uptake in rvZIP8 cells. CdCl2 was added to make a final Cd concentration of 0.25 mM; the competing metal cations (in chloride, or nitrate anion) at concentrations of 0, 1,
4 or 16 mM were added at the same time as Cd, and the cells were incubated at 37°C for 20 min, following which Cd accumulation was determined. Because Pb2+ precipitates in chloride-rich medium, we tested Pb2+ as the acetate salt, and its uptake medium was gluconate-HBSS. The Ag1+ ion (no effect on Cd uptake; data not shown) likewise precipitates with chloride and was also studied in gluconate-HBSS. All the other metals were chloride salts, and these studies were carried out in regular HBSS containing Cl–. Mn2+ was significantly (P = 0.0007) inhibitory at 1 mM compared with 0 mM Mn2+ added, and significantly (P = 0.0007) inhibitory at 16 mM compared with 4 mM Mn2+ added. Hg2+ was significantly (P = 0.0305) inhibitory at 1 mM compared with 0.25 mM Cd added; inhibitory trends seen with Cs2+ , Cu2+ , Zn2+ and Pb2+ were not statistically significant (P >0.05). These 2-tailed P values were calculated from Student’s t-test with four degrees of freedom.
Fig 3. Comparison of the kinetics of Cd (A) and Mn (B) uptake in rvZIP8 cells. The cells were incubated
(with the indicated concentration of cation) for 20 min at 37°C.
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Figure 4. Cell killing by 32-h exposure to Cd (A), Mn2+ (B), Hg2+ (C) or Zn2+ (D) in rvZIP8 versus rvLUC cells. Panels A, B, and C are semi-log plots, whereas D is a linear plot. Asterisks denote significant (P <0.05) differences between rvZIP8 and rvLUC––at the indicated concentration of the indicated cation (Student’s t- test). Pb2+ toxicity could not be done, because chloride-rich medium precipitates Pb2+, and the gluconate solution is perishable in less than 32 h at 37°C.
Figure 5. Effects of Na+ (A), K+ (B), or Cl– (C) substitution on Cd uptake in rvZIP8 and rvLUC cells.
NMDG+, N-methyl-D-glucamine. In all cases, cells were incubated with 0.25 mM Cd, containing
109 radiolabeled CdCl2, for 20 min at 37°C. The closed bars are not significantly (P >0.05) different from one another in each of the three panels (ANOVA), and the stippled bars are not significantly (P >0.05) different from one another in each of the three panels (ANOVA).
- Figure 6. Dependence of Cd uptake on bicarbonate in rvZIP8 cells. A, Cd uptake as a function of HCO3 concentration in HBSS. B, percent inhibition of Cd uptake as a function of increasing amounts of 4,4'-
- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a well known competitive inhibitor of HCO3 transporters.
The DIDs solution was prepared fresh in DMSO and added to the uptake medium 30 min before addition of
109 - - the CdCl2. For HBSS (~4 mM), the medium contained 4.17 mM HCO3 . For HCO3 -depleted HBSS (~0
– mM), the medium contained nominal amounts (171 mM) of HCO3 . In all cases, cells were incubated with
109 0.25 mM Cd, containing radiolabeled CdCl2, for 20 min at 37°C. *, mean is significantly (P >0.05)
– different from the mean when no DIDS was present at 4 mM HCO3 (Student’s t-test). **, mean is
– significantly (P >0.005) different from the mean when no DIDS was present at 171 mM HCO3 (Student’s t- test).
Figure 7. Western immunoblot of control, ZIP4ha, and ZIP8ha proteins, with or without treatment with
PNGase F, which cleaves glycoproteins. Lane-loading was confirmed by the protein b-actin.
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Figure 8. Z-stack confocal microscopy of MDCK cells, showing that NBC1_GFP transporter is basolateral
(left), whereas ZIP8ha (left and right) and PNA-lectin (right) are apical. Expression vectors carrying the indicated transporter proteins were transiently transfected into cells. After 2 days, cells were fixed, blocked and incubated with primary anti-ha antibody, then the secondary fluorescence antibody. Stained cells were examined under a Zeiss 510 laser-scanning confocal microscope. X-Y and Z denote the three axes examined.
NBC1, sodium bicarbonate-1 transporter. GFP, green fluorescent protein. ha, hemagglutinin C-terminal tag.
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- 74 -
- 75 -
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Table 1. Composition of Hank’s Balanced Salt Solution (HBSS)
Molarity (mM) in HBSS + + + + + – COMPONENTS Normal Na replaced with K Na replaced with NMDG K adjusted HCO3 adjusted D-Glucose 5.56 5.56 5.56 5.56 5.56 (Dextrose)
CaCl2 1.26 1.26 1.26 1.26 1.26
MgCl2 0.493 0.493 0.493 0.493 0.493
MgSO4 0.407 0.407 0.407 0.407 0.407 KCl 5.33 143.26 5.33 varied 5.33 NaCl 137.93 – – varied varied (NMDG+)Cl – – 137.93 – –
NaHCO3 4.17 – – 4.17 varied
KHCO3 – 4.17 4.17 – –
Na2HPO4 0.338 – –- 0.338 0.338
K2HPO4 – 0.338 0.338 – –
NaH2PO4 0.441 – – 0.441 0.441
KH2PO4 – 0.441 0.441 – –
COMPONENTS Glu– D-Glucose 5.56 (Dextrose) – Ca(Glu )2 1.26 – Mg(Glu )2 0.493
MgSO4 0.407 K(Glu–) 5.33 Na(Glu–) 137.93
NaHCO3 4.17
Na2HPO4 0.338
NaH2PO4 0.441
Where indicated, transport studies were conducted in HBSS (InVitrogen), which lacks Ca2+ and
2+ Mg . The standard HBSS that we made was modified to contain 1.26 mM CaCl2, 0.493 mM MgCl2,
0.407 mM MgSO4, and 4.17 mM NaHCO3 (2nd column). Adjustments were made to maintain these ion concentrations, using stock solutions of the respective salt. To determine anion and cation requirements for ZIP8 transport, the following modifications were made to the standard HBSS. For
+ + + 3- - - + Na -free K -HBSS (3rd column), Na salts of PO4 , HCO3 , and Cl were replaced with K salts.
For Na+-free NMDG+-HBSS (4th column), NaCl was replaced with NMDG chloride, and the Na+
- 3- + + salts of HCO3 and PO4 were replaced with K salts. For salt solutions in which K was varied (5th
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- column), KCl was replaced with NaCl. For salt solutions in which HCO3 was varied (last column),
- - NaHCO3 was replaced with NaCl. For Cl -free HBSS, Cl in all salts was replaced by gluconate
+ – – salts (last two lower columns). NMDG , N-methyl-D-glucamine ion. Glu , gluconate ion (C6O7H11) .
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Chapter V. Cloning and Initial Characterization of Mouse ZIP14A
and ZIP14B transporters
Abstract:
Cadmium (Cd) is a toxic trace metal widely distributed. Unfortunately, little is known about its molecular uptake by mammals. The involvement of ZIP (Zrt-, Irt-like proteins) in Cd uptake by mammals was first reported as a result of searching for the Cdm locus in inbred mouse strains
(Dalton et al, 2000; 2005). Expression of recombinant ZIP8 protein in cultured mouse fetal fibroblasts (MFFs) leads to as much as a 10-fold increase in the rate of intracellular Cd accumulation. Out of 14 ZIP proteins identified in the human and mouse genomes, ZIP14 protein is the one evolutionarily most closely related to the ZIP8 protein: they have a similar number of amino acids and the highest percent identity; both genes have the same intron-exon structure. In the following study, we identified 3 alternatively-spliced ZIP14 transcripts, with two of them displaying translation of fully functional proteins –designated as ZIP14A and ZIP14B. Using MFF cells as the expression system, we demonstrated that, similar to the ZIP8 protein, ZIP14 proteins have a high affinity for Cd and Mn. Expression of either of the two ZIP14 proteins in MFF cells greatly sensitized the cells to Cd killing (20-fold decrease of ED50) and Mn toxicity (3-fold decrease).
Consistent with their “influxer” role, ZIP14 proteins are localized to the plasma membrane of MFF cells, as demonstrated by Western blot and immunofluorescence microscopy. Furthermore, they were found to be localized to the apical side of polarized MDCK epithelial cells, implicating important roles in Cd absorption and deposition.
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(Introduction)
Cadmium (Cd, Cd2+) is a widely distributed toxic trace metal. It is found in industrial applications, in fertilizers, and in food products. Unlike most metals, Cd use began fairly recently with its large-scale application dating from the 1940s (Stoeppler, 1991). The toxicity of Cd administered in mammals varies markedly, depending on the dose and the duration of the exposure
(Zalups and Ahmad, 2003). Chronic exposure to low doses of Cd results predominantly in nephropathy, whereas acute exposure to large doses can result in damage to numerous tissues, including gastrointestinal tract, lung, liver, CNS, bone, ovary, placenta, and testis.
Even though many studies have been performed over the years, we still know very little about the molecular mechanisms of Cd transport in mammalian animals. Cd is a non-essential metal. Its environmental concentration has risen up only since industrialization; since then, Cd has posed a significant threat to human beings as an environmental pollutant. Thus, Cd-transporting proteins in all likelihood should have been initially “designed” by Mother Nature to transport other essential metal ions. For example, calcium channels and SLC11A2 (NRAMP2, DMT1), a proton-coupled divalent metal transporter with preference for iron, have been implicated in Cd uptake and toxicity in mammals.
The involvement of the ZIP8 protein (Zrt-, Irt-like protein 8; Irt and Zrt are the first members of this transporter family to have been identified) in Cd uptake by mammals was first reported as a result of searching for the Cdm locus (Dalton et al., 2005). In our previous study, ZIP8 expression in cultured MFF cells (mouse fetal fibroblasts cells) was found to lead to a 5- to 10- fold increase in the rate of Cd intracellular accumulation, and a more than 20-fold increase in the sensitivity to Cd-
- induced cell death. This Cd uptake is absolutely dependent on the presence of bicarbonate (HCO3 ).
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Radioactive Mn2+ uptake study shows that ZIP8 has a fairly high affinity for Mn2+. ZIP8 is a glycosylated protein which is localized to the apical side of polarized MDCK cells.
The official name for the ZIP protein family in human or mouse is the SLC39 family (solute- carrier super-family 39). There are 14 ZIP transporters encoded by the human and mouse genomes.
These protein members are expressed quite widely in human/mouse tissues, with distinctly different tissue expression patterns, and different, yet overlapping, substrate spectra. In plants, whereas IRT1 and TcZNT1 were found to be able to transport Cd, many other ZIP members are incapable of transporting this metal (Hall and Williams, 2003). Unfortunately, very few data have been collected regarding the manner in which Cd is handled by mammalian ZIP transporters.
To further assess the role of mammalian ZIP transporters in Cd transport, and to identify additional proteins that are capable of transporting Cd, we conducted the following study. We demonstrated that the ZIP14 protein(s) was evolutionarily the most closely related protein to ZIP8 in the mammalian SLC39 family. Therefore, we cloned the ZIP14 cDNA and expressed this cDNA in a cell culture system. Three alternatively spliced transcripts were found, with two coding for full- length, functional ZIP14 proteins. These were named ZIP14A and ZIP14B, corresponding to alternative exons 4 encoding the two different proteins. We also demonstrated that, similar to ZIP8,
ZIP14 proteins had a high affinity for Cd and greatly sensitized cells to Cd toxicity. Furthermore,
ZIP14 proteins were found to be glycosylated, and to be localized to the plasma membrane of non- polarized MFF cells and the apical membrane of polarized MDCK cells, implicating important roles in Cd absorption and deposition.
Materials and Methods:
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Bioinformatic tools. Protein sequences and their respective ZIP domain sequences were obtained from the NCBI website (www.ncbi.nlm.nih.gov).
ClustalW (http://www.ebi.ac.uk/clustalw/index.html) was used to construct a multiple alignment and a phylogenetic tree on human and mouse ZIP proteins (only the ZIP domain sequence of each protein was used to construct this tree).
To assess the topology and transmembrane (TM) domain sequences of ZIP proteins, each protein sequence was submitted to the online program MINNOU (membrane protein identification without explicit use of hydropathy profiles and alignments) at http://minnou.cchmc.org/ to detect the putative TM regions (Cao et al., 2006). These TM domain sequences were cut from each protein, and an artificial sequence was thus compiled for that protein. Percent identity was assessed, based on the pairwise alignment of these artificial sequences.
Cloning of the ZIP14 and ZIP4 cDNAs. Similar to the ZIP8 cloning process, as described in
Dalton et al in 2005, oligo-dT-primed reverse transcription was carried out on C57BL/6J mouse total testicular RNA. Primers were designed to PCR the coding sequence of the mouse ZIP14 cDNA
(from the NCBI database). The PCR products were then cloned into the Bam HI and Apa I sites of a retroviral vector pRevTRE (Invitrogen; Carlsbad, CA). The ZIP4 coding sequence was cloned from a plasmid kindly donated by Dr. Glenn Andrews (Dufner-Beattie et al, 2003). ZIP4 was cloned into the Bam HI and Sal I sites of pRevTRE. The ZIP8 coding sequence was cloned into the Bam HI and
Cla I sites of the pRevTRE vector (Dalton et al, 2005). A consensus Kozak sequence was included at the start-site of all three cDNAs for efficient expression. Furthermore, a hemagglutinin (ha) tag sequence was inserted, in-frame, right before the termination codon of each cDNA’s coding sequence.
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Delivery of the ZIP14 and ZIP4 cDNAs into MFF cells. The pRevTet-off vector (modified from the original Invitrogen vector by replacing G418 resistance gene with puromycin resistance gene) (Bergwitz et al., 2000; Dalton et al., 2005) was used to infect an immortalized mouse fetal fibroblast (MFF) line, and the resulting cells (designated as Tet-off cells) were selected by puromycin resistance at 3 µg/ml. The expression plasmids pRevTRE-ZIPs (the construction process was described above) were packaged respectively into replication-incompetent retroviral particles by 293GPG packaging cells (Solis et al., 2002), and the supernatant fractions were used to infect the Tet-off cell line to generate the rvLUC, rvZIP14A, or rvZIP14B, and rvZIP4 cell lines
(“HA” is omitted from the names of these ZIP cell lines for the purpose of simplicity). These cell lines were then selected by hygromycin resistance at 400 mg/ml.
Immunoblot Analysis of ZIP14 protein. As described by He et al, in 2006.
Measuring 109Cd/65Zn/54Mn Uptake. As described by He et al, in 2006.
Immunofluorescence Labeling and Confocal Microscopy. For immunofluorescence analysis of MFF cells, see Dalton et al, in 2005. For immunofluorescence analysis of MDCK cells (Z stack analysis), see He et al, in 2006.
Northern Hybridization Survey of ZIP8 and ZIP14 mRNAs in Mouse Tissues. Tissues from
B6 and D2 mice were harvested and frozen in liquid N2 until used. Total RNA was extracted, size- separated on agarose gels, and transferred to nylon membranes. The membranes were blocked and hybridized with either ZIP8 or ZIP14 cRNA probes. These probes were transcribed from two linearized plasmids respectively: the ZIP8 or ZIP14B open reading frame in pBluescript. The plasmids were linearized with Bam HI, and T3 RNA polymerase was used to synthesize a cRNA probe from the full-length ZIP8 open reading frame of 1,386 bp, and ZIP14B open reading frame of
1,487 bp.
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Statistical Analysis. As described by Dalton et al, in 2005 and He et al, in 2006.
Experimental Results:
ZIP14 and ZIP8 Are Evolutionarily Most Closely Related. In human and mouse, the official name for the ZIP protein family is SLC39. A protein gets assigned to a specific SLC family if it has at least 20-25% amino-acid sequence identity to other members of that family (Hediger et al., 2004).
Database analyses have shown that there are 14 ZIP transporters encoded by the human and mouse genomes (Eide, 2004). These protein members are highly related with respect to their function and evolution, and they are expressed widely in human and mouse tissues, with different tissue expression patterns. In order to assess the evolutionary relationship and the structure/function relatedness, a multiple alignment (using Clustal W) and a phylogenetic tree (Fig.1A) were constructed using human and mouse ZIP protein sequences in their respective ZIP domain regions.
There are two reasons why all 14 ZIP members must have been present before the divergence of human and mouse. First, the human and mouse genomes have the same number of SLC39 genes.
Second, the orthologous gene pairs between the two species are highly conserved (for example, the percent identity in ZIP domain sequences between mouse and human is 96% for ZIP8, 92% for
ZIP14).
ZIP8 and ZIP14 form a distinct branch in the phylogenetic tree, indicating that they are evolutionarily most closely related. In fact, it is most likely they have diverged from the other 12
ZIP members more than 400 million years ago. It is also necessary to point out that ZIP14 and ZIP8 proteins have somewhat similar lengths: mouse ZIP14 has 489 amino acids, and mouse ZIP8 has
462 amino acids (Fig.1C). In contrast, mouse ZIP4 has 660 amino acids, and mouse ZIP1 has 570 amino acids. Pairwise alignment (of two proteins in their ZIP-domain regions only) confirms this
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observation: of 14 mouse ZIP proteins, ZIP14 shares the highest percent identity – 73% – with ZIP8.
In contrast, ZIP4 shares 43% percent identity with ZIP8, and ZIP1 shares 16% percent identity with
ZIP8. ZIP14 also has a similar gene structure to ZIP8 (Tab. 1): they have the same number of exons, each exon has very similar, if not identical, lengths; and the exon-intron junctions are highly conserved (data not shown). Across the genomic regions of the Slc39a14 and Slc39a8 genes, highest homology is found in the coding exons, then the non-coding exons, and very low homology is found in the introns (data not shown). This is what is normally seen for all protein-coding genes.
Thus, if we want to find additional ZIP proteins that might be capable of transporting Cd, it is reasonable to extend our study from ZIP8 to the ZIP14 protein. In contrast, ZIP4 is an established
Zn transporter, moderately related to ZIP8, and its role in Cd uptake has not been studied. It would also be worthwhile to study ZIP4 protein in Cd uptake, to see whether the Cd uptake capability is a generalized property in the mammalian ZIP protein family, or a property only limited to the
ZIP14/ZIP8 branch of this family.
Cloning of Mouse ZIP14 cDNA and Identification of ZIP14 Alternatively-Spliced Forms.
During the cloning process, 3 alternatively-spliced forms of ZIP14 transcripts were identified.
Transcripts ZIP14A, ZIP14B and ZIP14AB (Fig.1B), have exon 4A, exon 4B, or both 4A and 4B, respectively. Exons 4A and 4B are both 170 bp long and share 67 % identity. Transcripts of ZIP14A and ZIP14B encode two different proteins of the same length (489 amino acids). These two proteins are only slightly different in a 44-amino acid region. Based on bioinformatics predictions, this
“slightly different region” is located in the intracellular loop between TM domain I and II, and it does not change the topology of this transporter protein. Transcript ZIP14AB has a disrupted reading frame and would code for a truncated protein with only 157 amino acids. This alternative splicing pattern of ZIP14 mRNA was reported (Liuzzi et al., 2005), but wrongly attributed to exon 5.
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It is interesting to note that exon 4 of ZIP8 is also 170 bp, with similar homology (67% identity) to exon 4A or exon 4B of ZIP14 (data not shown). It is interesting to note that exon 4 of Slc39a8 is also 170 bp, with similar homology (67% identity) to exon 4A or exon 4B of Slc39a14 (data not shown). This finding suggests that the divergence of SLC39A8 and SLC39A14 was due to an ancient gene duplication event; and, after this gene duplication, an exon 4 duplication event occurred for
SLC39A14.
The ZIP14 sequence used to construct the phylogenetic tree was derived from the reference sequence NM_144808 of the NCBI nucleotide database. This was confirmed to be ZIP14B in our nomenclature. The phylogenetic tree analysis would be the same if ZIP14A sequence was used instead.
Western Analysis of ZIP-Overexpressing Cells. Infected MFF cells were selected in culture medium containing 400 mg/ml hygromycin and 3 mg/ml puromycin. The expression profile of ZIP transporters was examined by Western analysis using anti-ha antibody (Fig.2). Similar to the
Western immunoblot of ZIP8 (Dalton et al., 2005), the ZIP14A, ZIP14B and ZIP4 proteins were found only in the membrane fraction (including plasma membrane and vesicular membrane) of the cells, and not detected in the cytosolic fraction (data not shown).
Glycosylation is one of the most common post-translational modifications of membrane proteins and secretory proteins in eukaryotic cells. There are two potential N-linked glycosylation sites in the
ZIP8 protein sequence, Asn-40 and Asn-88; with PNGase F treatment, ZIP8 protein has been demonstrated to be glycosylated (He et al, 2006). Following the same rationale and technique,
ZIP14 proteins were found to have 3 potential glycosylation sites: Asn-75, Asn-85, and Asn-100.
All sites are in the extracellular N-terminus. After PNGase F treatment, there was a significant decrease in the molecular weight of the ZIP14 protein band from about 80 kD to about 50 kD,
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which is close to the calculated molecular weight of ZIP14 proteins. Similarly, the ZIP4 protein was also shown to be glycosylated. The native ZIP4 protein had two very close bands of about 70 kD, consistent with previous findings (Wang et al., 2004a). The heterogeneity of glycosylated ZIP protein bands may be due to the heterogeneous nature of the oligosaccharide chains attached to the protein, as well as various degrees of saturation at the available glycosylation sites. Based on the intensity of the bands, we can also see that the expression levels of different ZIP proteins were similar in our system.
109Cd /54Mn/65Zn uptake By ZIP Proteins. Because ZIP8 had previously been shown to take up Cd and Mn efficiently, 109Cd and 54Mn were the first two cations to test for substrate specificity of the ZIP14 transporters. Fig. 3 shows that, at the concentration being examined––0.25 mM for Cd or Mn––rvZIP14A and rvZIP14B cells internalize 109Cd and 54Mn several-fold faster than the control rvLUC cells, with the relative order as: rvZIP14A > rvZIP14B >> rvLUC. Thus, ZIP14 proteins are good transporters in mediating Cd and Mn uptake.
ZIP4 protein, on the contrary, does not transport Cd at all (data not shown), at least at low micromolar levels of Cd. The protein expression levels of ZIP4, ZIP14A and ZIP14B transporters, as shown in the previous Western blot (Fig. 2), were not significantly different from one another.
Therefore, the above Cd uptake result also dismisses the concern that overexpression of any metal transporter could lead to the general elevation of Cd uptake.
65Zn uptake studies were also done (not shown). ZIP8, ZIP14A, ZIP14B and ZIP4 transporters can transport Zn at approximately the same rate –their overexpressing cells take up Zn twice as much as the control cells. This result is consistent with many previously reported Zn uptake studies on cultured cells transfected with ZIP1, 2, 3, 4, 5, 6, 7, 8, and 14 proteins. Some of these studies were done with radioactive Zn, and others were done using a Zn-responsive fluorescence dye.
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Therefore, we might tentatively conclude that Zn transport is a generalized feature for all ZIP family members, whereas not every ZIP transporter can transport Cd.
Toxicity to Various Metal Ions Mediated By ZIP14. The ZIP8 protein has fairly high affinity to Cd (Km = 0.62 mM) and Mn (Km = 2.2 mM); thus, the expression of ZIP8 protein was found to sensitize cells to Cd and Mn toxicity. Similarly, acute toxicity of Cd, Mn, as well as Zn, were tested in control rvLUC cells and all ZIP protein-expressing cells in a 32-hr time period. The MTT assay was used to measure cell survival.
The rvZIP8 and rvZIP14 cells were similar in their sensitivity patterns: they are extremely sensitive to the cytotoxic effects of Cd (the Cd toxicity curve shifted to the left >20-fold, as measured by ED50; Fig. 4A), and moderately sensitive to Mn toxicity (the Mn toxicity curve shifted to the left 2-fold; Fig. 4B). The sensitivity of rvZIP8 and rvZIP14 cells to Zn toxicity was indistinguishable from the control rvLUC cells (not shown). The ZIP4 protein did not sensitize the cells to any of these three metal ions.
The metal toxicity results are very consistent with the radiotracer uptake experiment. ZIP8 and
ZIP14 transporters take up Cd and Mn efficiently, thus leading to an elevation of the cells’ sensitivity to Cd and Mn.
Subcellular Localization of ZIP14 Proteins. Immunofluorescence was carried out using confocal microscopy to examine the subcellular localization of ZIP14 proteins in MFF cells (Fig.
5A). The images clearly show that, just as was seen for the ZIP8 protein (Dalton et al., 2005),
ZIP14A and ZIP14B proteins were both mainly localized to the plasma membrane of the MFF cells.
Uptake of Cd in vivo is a vectorial process, which happens in the epithelial cells of various tissues. Epithelial cells are polarized cells, with their apical side facing the lumen, and their basolateral side facing the basal membrane and tissues. Madin-Darby canine kidney (MDCK) cells,
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a DCT (distal convoluted tubule) polarized epithelial cell line, were chosen to further study ZIP14 subcellular localization. GFP-NBC1 is the fusion protein of green fluorescent protein and the
+ - Na /HCO3 cotransporter NBC1, and is localized to the basolateral membrane of MDCK cells (Li et al., 2004). Z-stack analysis (Fig. 5B) clearly shows that ZIP14 proteins are apically localized. Thus, our result implies that –similar to the ZIP8 protein –the ZIP14 protein also functions to take up endogenous or exogenous substrates from the lumen into tissues or organs where they are expressed.
Discussion:
The ZIP family has grown to more than 575 members, including proteins in bacteria, nematodes, insects, and mammals (from Pfam database of Sanger Institute, 05/04/2006: http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF02535). Many ZIP transporters have been implicated in Zn, Fe, and Mn transport in plants. The ZIP transporters transport metal-ion substrates across cellular membranes into the cytoplasm. Whereas many ZIP members are involved in the uptake of metal ions across the plasma membrane, some might be involved in the efflux of metals from intracellular compartments into the cytoplasm (Huang et al., 2005).
Our previous study showed that ZIP8 has an extremely high affinity for Cd. The Km for Cd uptake by ZIP8 is only 0.62 mM in our cell culture system, about half the Km of DMT1 (SLC11A2) as determined in Xenopus oocytes. The study presented here shows that ZIP14, the ZIP protein most closely related to ZIP8, can also transport Cd at high velocity. Furthermore, expression of ZIP14 greatly sensitized the cells to Cd-mediated toxicity, to the same degree as ZIP8. It is possible that the Cd uptake ability is not shared by every member in the ZIP protein family, since ZIP4, a member belonging to LIV-1 subgroup and moderately related to ZIP8/ZIP14 proteins, did not take up Cd at concentrations being examined, nor did it sensitize the cells to Cd toxicity. We have caught
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at least two culprits for molecular “smuggling”of Cd; the roles of the other remaining eleven ZIP transporters in mediating Cd transport and toxicity still need to be examined.
It will be interesting to understand why ZIP8 and ZIP14 proteins have such a high affinity for
Cd. The most straightforward hypothesis would be that they share critical amino acids or signature sequences that determine the substrate metal specificity, since the topologies of all mammalian ZIP proteins are highly similar. Consistent with this hypothesis, determinants of substrate specificity have been mapped to the extracellular loop between TM II and III in the Irt1 transporter (Rogers et al., 2000), and the conserved histidyl, serine, and glycyl residues in TM IV are essential for uptake activity of all substrates. Similar mutational experiments could be carried out for ZIP8 and ZIP14 proteins to identify sequence feature(s) that cause Cd specificity.
Cd is not an essential metal and its risk to human populations only rose significantly with industrialization. Thus, it must be a “hitchhiker” for all the transporters that it is using. What, then, are the physiological substrates of ZIP14? Our previous study has shown that ZIP8 can take up Mn at high velocity and fairly high affinity. Similar to this result, we showed here that ZIP14 proteins can take up Mn efficiently at 0.25 mM level, and the ZIP14-expressing cells have the same degree of elevated sensitivity to Mn toxicity as ZIP8. Thus, it is very likely that Mn is also a physiological substrate for the ZIP14 proteins. Surely, their capability in mediating Mn uptake does not negate their role in regulating Zn uptake and homeostasis. In fact, ZIP14 has already been shown to play a role as a Zn transporter during the early stages of adipogenesis (Tominaga et al., 2005).
The significance of the alternatively spliced exon 4 gene products, leading to functional ZIP14A and ZIP14B transporters, still remains unknown.
The confocal analysis shows that ZIP8 (He et al, 2006), ZIP14 (Fig.5B) and ZIP4 (Wang et al.,
2004b) proteins were all localized mainly to the plasma membrane. These observations are in
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agreement with many reports that ZIP proteins are mainly localized to the plasma membrane. More specifically, using cultured MDCK cells as a model system, we showed that expressed recombinant
ZIP8 and ZIP14 proteins are principally localized to the apical side of these polarized epithelial cells (Fig. 5B). This implies that, the vectorial process of Cd (Mn, Zn as well) transport by ZIP8 and
ZIP14 proteins are from the lumen side to the tissue side in the epithelial cells. This is consistent with the reported vectorial process of ZIP1, ZIP2, ZIP3 and ZIP4. The ZIP5 transporter also brings substrate ions into the epithelial cells; however, it is localized on the basolateral side of the epithelial cells. The in vivo role of ZIP5 is thus intriguing and significantly different from all the other ZIP transporters that have been characterized to date.
ZIP8 and ZIP14 are very similar in their substrate specificity and their subcellular localization.
This does not necessarily mean that their in vivo roles (either physiological or toxicological) are the same. They may have very different expression and regulation that are both tissue-specific and cell type-specific. For example, a tissue survey showed that ZIP8 mRNA is most abundant in the pancreas (Begum et al, 2002) and lung (Fig.6). ZIP14, however, has the highest mRNA level in small intestine (not shown), then kidney and liver (Fig.6). These tissue distribution patterns have important toxicological implications, since small intestine is the main absorption organ for oral Cd exposure, and lung is the main absorption organ for inhaled Cd. ZIP8 is also highly expressed in the endothelial cells of the testicular vasculature, and thus responsible for the sensitivity (in many inbred mouse strains) to Cd-induced testicular toxicity (Dalton et al., 2005).
With the more or less completion of the Human Genome Project and Mouse Genome project, relatively complete lists of protein-coding genes are now available. Based on sequence homology and domain structure, these genes can be grouped into superfamilies and families. Cd is not an essential metal; therefore, it must hitchhike on essential metal ion transporters. Thus, we can search
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for Cd-transporting proteins and study their structure and function in the context of all metal-ion transporter protein families, in a thorough and systematic way. There are four other SLC families that transport Zn and other essential divalent cations: the SLC11 proton-coupled metal ion transporter family, the SLC30 Zn effluxer, the SLC31 copper transporter family, and the SLC41
MgtE-like magnesium transporter family. Outside the SLC family, some ATPase pumps and ABC transporters can also transport metals as well. Cd could be a potential substrate for these metal transporters. Their in vitro and in vivo roles in mediating Cd uptake and toxicity need to be examined.
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Figure Legends
Figure 1A – Phylogenetic tree of mammalian ZIP proteins
Nearest neighbor-joining (NNJ) phylogenetic dendrogram, generated from the amino acid
sequences of the 14 members of mouse Slc39 and human SLC39 family. The mouse and human
39A14 and 39A8 members (in red) can be seen to be most closely related, in terms of
evolutionary divergence.
1B – Mouse ZIP14 alternatively spliced forms
Structural analysis of the mouse Slc39a14 gene (introns not drawn to scale), illustrating the
alternative splicing pattern for the two exons 4 (top). Differentially-spliced mRNAs, which
include exon 4a or exon 4b, give rise to ZIP14A and ZIP14B transcripts/proteins, respectively.
Similar to Slc39a8, this gene also has three alternatively spliced noncoding exons 1 (not shown).
Coding region is closed, 5’ and 3’ UTRs are open, rectangles.
1C – Alignment of ZIP14 and ZIP8 proteins
Boxes show the putative membrane localization signal and arrows indicate possible cleavage
sites. Potential glycosylation sites are indicated with *. Putative transmembrane regions are
underlined and denoted as TM.
Figure 2 – Western blot of ZIP proteins
Western immunoblot of control, ZIP14Aha, ZIP14Bha and ZIP4ha proteins, with or without
treatment with PNGase F, which cleaves glycoproteins. Lane-loading was confirmed by the
protein b-actin.
Figure 3 – Cd//Mn uptake by rvZIP cells
Uptake of radioactive Cd (A) and Mn (B) by various types of cells in HBSS. Cells were
exposed to 0.25 mM Cd or Mn for 20 min at 37º C.
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Figure 4 – Cd/Mn in vitro toxicity mediated by rvZIP cells
Cells were cultured for 32 h in the indicated concentrations of Cd (A) or Mn (B), and cell death
was monitored using the MTT assay.
Figure 5A – ZIP protein membrane localization in MFF cells
Subcellular localization of the various ZIPha proteins. Cells were fixed, permeabilized, and
incubated with a rabbit anti-hemagglutinin (ha) antibody. Cells were then washed and incubated
with a goat anti-rabbit FITC-conjugated antibody. Cells were counterstained with propidium
iodide (PI) to visualize nuclei. Fluorescence microscopy was used to detect PI and FITC. FITC,
fluorescein isothiocyanate.
Figure 5B – Localization of the ZIP proteins to the apical surface in MDCK cells
Z-stack confocal microscopy of MDCK cells, showing that the NBC1_GFP transporter is
basolateral, whereas ZIPha proteins are apical. Expression vectors carrying the various proteins
were transiently transfected into the cells with Lipofectamine 2000. After 2 days, cells were
fixed, permeabilized, blocked, and then incubated with primary anti-ha antibody, followed by
the secondary fluorescence antibody. The immunostained cover slips were examined under a
Zeiss 510 laser scanning confocal microscope. GFP green fluorescent protein. ha, the
hemagglutinin C-terminal tag. X-Y and Z denote the three axes examined.
Figure 6 – Northern Survey of ZIP8 and ZIP14 mRNA in Mouse Tissues
B6 and D2 represent two mouse strains where tissues were taken from. Ethidium Bromide (EB)
staining were RNA loading controls.
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Figure 1A.
Figure 1B.
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Figure 1C.
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Figure 2A.
Figure 2B.
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Figure 3A.
Figure 3B.
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Figure 4.
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Figure 5A.
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Figure 5B
Figure 6.
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Table 1 – Comparison of the Slc39a8 and Slc39a14 gene structures
Mouse Slc39a8 Mouse Slc39a14
Exon Intron Exon Intron
1c 256 1,486 1c 139 32,338 1b 146 632 1b 110 29,381 1a 37 156 1a 132 15,128 2 451 26,094 2 280 2,037 3 163 6,810 3 187 604 4 170 1,408 4a 170 4,688 4b 170 2,625 5 123 465 5 120 584 6 171 25,966 6 189 117 7 208 197 7 208 923 8 185 2,018 8 185 1,837 9 1,586 9 516
For mouse Slc39a8, the longest mRNA length is 3,313; the gene spans 67,757 bp. For mouse Slc39a14, the longest mRNA length is 2,164 bp; the gene spans 45,122 bp.
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Chapter VI. Conclusions and Speculations
Conclusions:
1. The Slc39a8 gene is the Cdm locus, encoding the gene product (ZIP8) that determines
testicular sensitivity to Cd toxicity; sensitive strains of mice show high expression of ZIP8
in the endothelial cells of the testicular vasculature, and resistant strains of mice show
negligible amounts of expression in these endothelial cells.
2. ZIP8 is a rogue Cd transporter; it is located on the apical side of epithelial cells; under
2+ - physiological conditions, ZIP8 is likely to be a Mn /HCO3 symporter––although Zn
transport cannot be ruled out.
3. ZIP14 is evolutionarily the closest member to the ZIP8 transporter; it has 3 alternatively
spliced transcripts and 2 functional proteins; ZIP14A and ZIP14B also have a high affinity
for Cd and Mn; they are also localized to the apical side of epithelial cells.
Speculations:
Identification of the causal mutation and haplotype. The study in Chapter II demonstrated that the Slc39a8 gene is the Cdm locus. However, the causal mutation for the resistance phenotype of CITN has not yet been identified. A 170-kb BAC clone containing the Slc39a8 gene’s sensitive allele (isolated from a BAC library derived from the 129/SvJ mouse strain) has been identified in our laboratory. This BAC contains a 85-kb upstream region, the 67-kb region containing all introns and exons, and a 18-kb downstream region. Transgenic mice carrying this BAC have been generated and bred onto the B6 (resistant) mouse background. To our great excitement, this
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transgenic mouse line showed great sensitivity to CITN (data not shown). In other words, the presence of sensitive Slc39a8 transgene (129/SvJ allele) reversed the resistance phenotype of the B6 mouse. This experimental result further corroborates our conclusion that the Slc39a8 gene is the
Cdm gene. It also shows that that the causal mutation leading to resistance in the B6 mouse strain must be located in this 170-kb region defined by the transgenic BAC clone.
From the SNP database of Celera, we know that there are 136 SNP differences between D2 and
B6 mouse strain in this 170-kb BAC transgenic region. Any one of these SNPs could be the potential causal SNP, which leads to the differential expression of ZIP8 in the vascular endothelial cells of the mouse testis. And we have to take into consideration that the SNP database of Celera is not yet complete. It would seem to be a formidable job to find the causal mutation. Nevertheless, there are still some possible approaches to identify the culprit.
1. Bioinformatic analysis of the response elements in the 170-kb region
From our data, we know that the genetic difference leading to the differential ZIP8 expression in testicular endothelial cells most likely reflects a transcriptional difference. The basic rationale of this strategy is to use online databases for transcription-factor-binding sites, such as TRANSFAC
(http://www.gene-regulation.com/index.html), to identify all possible response elements in the 170- kb region. We can then overlay the D2/B6 SNPs on these response elements to find out those SNPs that could potentially alter the canonical sequence and, thus, the function of the response element.
Once we can reduce the potential SNPs to a small enough number, we can do a refined promoter- reporter analysis, DNase protection assays, EMSA assays, etc. to further identify the specific altered response element between D2 and B6, and thus to identify the causal mutation.
This approach is straightforward, but the caveats are many. First, the Celera SNP database is not complete; it does not cover all the SNP differences between D2 and B6 mouse. Second, the
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transcriptional database is in an even more primitive form, far from being thorough and accurate.
We still know very little about response elements or locus-control regions for tissue-specific gene expression. Third, after being “sifted” by this approach, half of the 136 SNPs were found to potentially change one or another transcription-factor binding site (data not shown). Therefore, this approach does not help very much in reducing the number of candidate SNPs.
2. Chimeric BAC transgenesis
With the recent advance in ET-recombineering (Muyrers et al., 2001), we can modify long stretches of DNA sequences, such as those in a BAC clone, with ease. The rationale of this approach would be to replace part of our transgenic BAC clone (from 129/SvJ strain allele) with the equivalent part of the resistant B6 allele sequence; then we can make a transgenic mouse line with this chimeric BAC clone. If the transgenic mouse (bred back onto B6 mouse background) is sensitive, then the causal mutation would exist in the part that is not swapped by the B6 allele sequence; if the transgenic mouse is resistant, the causal mutation exists in the part that has been swapped by the B6 allele sequence. In this way, we can narrow down the region containing the causal mutation. This would be the most reliable approach. The downside of this approach, however, is that the experiments would require much time, money and effort.
3. Haplotype block analysis
This approach stems from one analysis result of the Celera mouse SNP database. Fig.1 lists all the SNPs detected by Celera in the 170- kb region between several mouse strains. We can see clearly that B6 and A/J –two resistant mouse lines, share the same nucleotide in every SNP location detected by Celera, which implies that the DNA sequences in this whole region are identical between B6 and A/J strains (both being Cd-resistant strains). This is very intriguing, considering the many historical meiotic events that have happened in the establishment and divergence of these two
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mouse lines; obviously, no recombination events have occurred in this DNA region that could have disrupted the haplotype block. Considering the limited numbers of inbred-strain-founder populations (Beck et al., 2000), it is very likely that all resistant laboratory mouse strains inherited the same founder allele in the Slc39a8 gene region. This is not the case for the sensitive D2 and 129 alleles: there are many different nucleotides between them; therefore, they probably do not share the same ancient allele; these different sensitive alleles were formed either before––most likely––or after, the establishment of the laboratory inbred mouse strains. This mosaic haplotype pattern in the genome of inbred mouse strains has been examined in a handful of recent literature reports (Park et al., 2003; Liao et al., 2004; Yalcin et al., 2004; Zhang et al., 2005).
This analysis result inspired an approach: we can find the smallest haplotype block in this region that is shared by all the resistant inbred mouse strains. This smallest haplotype block defines the causal mutation-containing region. Actually, there is one successful report of finding a causal mutation using this haplotype block-phenotype association approach (Liao et al., 2004). Technically speaking, first, we can select 10-20 SNP markers (between D2 and B6) spanning this 170 kb-region and genotype all resistant inbred mouse strains; the smallest resistant haplotype block would contain the causal mutation leading to the resistance phenotype. Next, we can employ all the Celera SNP markers in the region, refined by the first step, and genotype several or even all sensitive strains to find out the haplotype structures. From all of the constructed haplotype blocks in the sensitive alleles, those that are not shared by the resistant mouse allele should contain the causal mutation. In this way, the causal mutation-containing region can be further refined.
These three approaches are not mutually exclusive. In the best scenario, we can first use haplotype block analysis to narrow down the mutation to a very small region; then use bioinformatic tools to identify all altered response elements between B6 and D2 alleles in the
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refined region; after further doing electromobility shift assays (EMSA), DNase protection assays, promoter-reporter studies, or other in vitro assays, the number of potential causal mutation- containing response elements can be further reduced; finally, BAC transgenesis could be carried out to test whether one or another SNP can cause the alteration of testicular sensitivity to Cd toxicity.
It is noteworthy to point out that the CITN phenotype can only be diagnosed in the intact mouse: either via testicular necrosis, or in situ hybridization to ZIP8 mRNA in the testis. There is no simple cell culture assay or in vitro assay that can test this phenotype. Thus, BAC-transgenic mouse lines, containing the only candidate SNP, would be the ultimate criterion in identifying the causal mutation.
Testicular-endothelium-specific regulatory element. There are three endothelial specific regulatory elements that have been reported to date. A transcriptional enhancer from the mouse
Mef2c (Myocyte Enhancer Factor 2C) gene is sufficient to direct gene expression to the vascular endothelium in transgenic embryos (De et al., 2004). This enhancer contains four perfect consensus
Ets transcription-factor-binding sites that are efficiently bound by the Ets-1 protein in vitro and are required for enhancer function in transgenic embryos. Another report came from the study of the Scl gene, which encodes a basic helix-loop-helix protein and plays a pivotal role in the normal development of both blood and endothelium. A bifunctional 5' enhancer (–3.8 element) was identified, which is both necessary and sufficient to target Scl gene expression to hematopoietic progenitors and endothelium (Gottgens et al., 2004). This enhancer contains conserved critical Ets sites, and is bound by Ets family transcription factors, including Fli-1 and Elf-1. In another report, the regulatory elements of the murine Flk-1 gene have been identified. It can mediate endothelium- specific expression of a LacZ reporter gene in transgenic mice (Kappel et al., 1999). Sequences within the 5'-flanking region of the Flk-1 gene, in combination with sequences located in the first
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intron, specifically target transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5'-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2 .
Testicular endothelium is different from the “regular” endothelium. Certain tissues, such as brain and testis, require a highly “protected” environment for their physiological functions. Thus, a biological barrier is required to prevent the free exchange of solutes between blood and tissue. For the blood-brain barrier (BBB), the barrier function is primarily achieved by the endothelium. For the blood-testis barrier (BTB), the first barrier is endothelium, impeding substances in the blood from getting into the interstitial space easily; then, the Sertoli cells and their inter-cell junctions further prevent the substances in the interstitial fluid from getting into the seminiferous tubules easily (Ploen and Setchell, 1992). In testis, the endothelium is continuous, not fenestrated, and has long junctional profiles; however, compared with brain endothelium, the testis endothelium still has a higher permeability (Holash et al., 1993). Barrier function is just one side of the story. The endothelial cells in brain versus testis have quite different sets of transporters from the regular endothelial cells (such as glucose transporters), maybe even from each other. This is a possible reason why ZIP8 has a highly tissue- and cell-type- specific expression pattern.
The sensitive ZIP8 allele product clearly has a testicular-endothelium-specific regulatory element that is functioning very well; in the Cd-resistant mouse, this element is mutated and becomes non-functional. To date, no such response element or locus-control region has been reported in testicular endothelial cells. If we can identify the causal mutation for the mouse strain resistance to CITN, we can subsequently identify this response element; in a further step, we can identify the transcriptional factor(s) that interact with this response element.
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The identification of the locus control element that confers highly specific testicular endothelial expression is of great theoretical and practical value. I shall name a few interesting applications here.
For example, an “expression vector” for testicular endothelial cell-specific transgene expression, can be constructed based on this knowledge. We can put the coding sequence of Egfp in the proper place. These manipulations can be easily achieved with the recent development of the ET- recombineering technique, even when the “expression vector” has to be a BAC clone. In this way,
Egfp should have the same tissue/cell expression pattern as the Slc39a8 gene does. This mouse line will help researchers easily determine and isolate the testicular endothelial cells in immunofluorescence studies. Similarly, b-Gal or other marker genes can be fused –to make transgenic mice that will be useful for immunohistochemistry or other purposes.
Besides being used for gene “knock-in”, this expression vector can also be used for gene
“knock-out”. For example, the Cre gene can be fused to this vector, and we can make a transgenic mouse line with Cre being expressed only in testicular endothelial cells. By breeding this mouse line with other gene-floxed mouse lines, conditional knockout mouse lines for any other gene can be generated (Nagy, 2000), in which the gene is only deleted in the testicular endothelial cells.
Therefore, this mouse line would be extremely helpful in studying the genes that are especially important in maintaining the BTB function and the transporting function of the testicular endothelium.
Mn transporter. Mn can exist in 11 different oxidation states between -3 to +7, with the +2 valence being the most prevalent in biological tissues. As an essential metal, Mn has many important functions (Gerber et al., 2002). Mn is a component of the mitochondrial enzymes pyruvate carboxylase, superoxide dismutase-2 (SOD2), glutamine synthetase, alkaline phosphatase, and arginase. Mn activates a large spectrum of enzymes. Mn-containing SOD2 has found particular
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attention because it participates in antioxidant activity and tumor defenses and may be increased or decreased in cancer cells. Mn is also essential for normal bone structure and the formation of mucopolysaccharides.
Human daily intake of Mn with food varies between 5.4 and 12.4 mg; a daily intake of 2–3 mg is considered to be adequate. The human body contains about 10–20 mg of Mn, of which 5–8 mg are turned over daily. Almost 80% of all Mn taken up is excreted via the bile and pancreatic fluids and appears in the feces. As noted earlier, ZIP8 expression is extremely high in pancreas (Begum et al., 2002); this finding is therefore consistent with the possible role of ZIP8 as a Mn transporter.
About half the Mn in the body is found in bones; the remainder is found in the active metabolic organs – liver, pancreas, pituitary gland, adrenal gland, and kidney. Although brain takes up less
Mn, it retains it tenaciously (half-life 34 d in rats). In plasma, approximately 80% of Mn is bound to b1-globulin and albumin, and a smaller fraction of Mn is bound to transferrin. When complexed with transferrin, Mn is exclusively present in the trivalent oxidation state (Aschner, 2000).
Mn deficiency has been observed in humans only very infrequently (National Research Council,
1989). It has been suggested that infant mortality rates are lower when the blood Mn levels are maintained at a certain level. In animals, experimental Mn deficiency results in a variety of symptoms, and the most prominent ones are those during pregnancy and early postnatal development (ATSDR and USPHS, 1992).
Chronic toxicity in humans mainly comes by way of occupational exposure, especially to the
CNS; this represents the predominant toxic effect of Mn for occupationally exposed populations
(Aschner, 2000). Mn readily crosses the blood-brain-barrier of the fetus, neonate and adult mammals. The brain normally contains only a small fraction of Mn. Excessive Mn in the brain will lead to toxicity. The clinical features of Mn neurotoxicity resemble those of idiopathic
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Parkinsonism, including bradykinesia, dystonia, disturbance of gait, fixed facial expression, and difficulty in speech. Dissimilarities between Mn toxicity and Parkinson disease include a less frequent resting tremor, more frequent dystonia, and failure to achieve a sustained therapeutic response to levodopa –in patients with Mn toxicity.
Very little is known about the molecular transport of Mn by mammals. There are studies indicating that transferrin, DMT1 and calcium channels might be involved in Mn uptake. Mn3+ is transported extracellularly as a transferrin complex. This complex binds to the transferrin receptor, which subsequently undergoes internalization within endosomal vesicles. The endosomal vesicles go through acidification, presumably releasing Mn3+ from the transferrin/transferring-receptor complex. The unbound Mn3+ need to be reduced in order to be transported across the endosomal membrane into the cell via the transporter DMT1 (Fleming et al., 1998). Mn can also be taken up directly into the cell by a transferrin receptor-independent mechanism requiring DMT1. DMT1 has very broad substrate specificity, including Fe2+, Mn2+, Cd2+, Cu2+, etc (Gunshin et al., 1997). Mn has a relatively high affinity for DMT1 and effectively competes with iron for binding to this transporter (Roth et al., 2002). There is an observation (Mena et al., 1969) that, just like Cd, intestinal absorption of Mn is enhanced by iron deficiency. In addition to cellular uptake by DMT1, there is also evidence in the literature for transport of Mn via voltage-regulated Ca2+ channels
(Lucaciu et al., 1997; Kannurpatti et al., 2000). Whichever mechanism is the predominant form for
Mn uptake is likely to reflect tissue- and cell-type-specificity. The in vivo physiological and toxicological importance and roles of these mechanisms are still unknown.
ZIP8 and ZIP14 have been identified so far, as having high affinity for Mn2+. This suggested that they might play important roles in mediating Mn uptake and maintaining Mn homeostasis in the intact animal. This would be an interesting field to study, and technically, we are quite prepared for
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this. First, it would be very helpful in studying the tissue- and cell-type-specific expression patterns of these two transporters. Then, the above-mentioned ZIP8 BAC-transgenic mice and ZIP8
(conventional versus conditional) knockout mice from this laboratory will be of great value for this field of study. We can put these mice on a Mn-sufficient or deficient diet, or challenge them with different regimens of high doses of Mn, so that we can compare the phenotypic changes of these mice with that of the wild-type mice. Radiotracer experiments can also be done on these mice, to compare the Mn distribution pattern with that in wild-type mice.
Another interesting experiment going on now is that we have been making a B6.D2-Slc39a8d congenic mouse line. It is being generated by repeated backcrosses into the B6 mouse background, with selection (at each round of breeding) of the presence of the D2 allele of the Slc39a8 gene. This mouse line would be useful in assessing the role of ZIP8 on mouse fertility, under normal condition or Mn-deficient condition. Such a congenic line would be identical, in theory, to the B6 mouse–– except for the Slc39a8d allele; comparison of the B6.D2-Slc39a8d mouse with the B6 wild-type mouse offers a much more rigorous study than simply a comparison of the Cd-sensitive D2 mouse with the Cd-resistant B6 mouse.
Apical versus Basolateral Localization of ZIP proteins. Correct subcellular localization is essential in maintaining the physiological functions of all proteins. Mutations in the subcellular localization signal can lead to human disease. For example, a form of familial hypercholesterolemia is caused by a mutated basolateral sorting signal in the LDL receptor (Koivisto et al., 2001).
Several AEZ-associated human missense mutations (G340D, L382P, G384R, G643R) have been identified, which cause failure of normal trafficking of the ZIP4 protein onto the intestinal cell surface at high levels (Wang et al., 2004a).
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ZIP1, ZIP2, ZIP3, and ZIP4 are located on the apical side of mouse enterocytes and mouse visceral yolk-sac epithelial cells. Consistent with these reports, we identified the apical localization of expressed recombinant mouse ZIP8 and ZIP14 proteins in MDCK cells. Very intriguingly, ZIP5 was found to be localized to the basolateral membrane in enterocytes, acinar cells, visceral endoderm cells (Dufner-Beattie et al., 2004), and cultured MDCK cells (Wang et al., 2004b). When mice were fed a Zn-deficient diet, ZIP4 protein was greatly enriched onto the cell surfaces; when mice were fed a Zn-adequate diet, ZIP4 protein quickly became internalized (Dufner-Beattie et al.,
2004). This protein trafficking pattern was demonstrated to be true in the MDCK cell system for the
ZIP1, ZIP2, ZIP3, and ZIP4 proteins. The ZIP5 protein functions in direct contrast to this trafficking pattern: ZIP5 internalizes upon Zn treatment and moves onto membrane when Zn gets depleted.
These results suggest that the function of ZIP5 is antagonistic to that of ZIP4 in the control of Zn homeostasis: ZIP4 acquires dietary Zn; ZIP5 removes Zn from the body, probably via the pancreas and intestine.
Generally speaking, whether a protein is transported to the apical or the basolateral plasma membrane should depend on the sorting signals located in the protein itself. ZIP proteins are fairly highly related to one another. ZIP4 and ZIP5 protein share 36% homology over their entire length and 50% homology in the ZIP domain. Furthermore, their predicted TM topology is similar: ZIP proteins have 6 to 8 TM domains; ZIP4 and ZIP5 have 8 TM domains. Thus, it is very tempting to speculate that ZIP1, ZIP2, ZIP3, ZIP4, ZIP8 and ZIP14 share some common sequence motif that determines their subcellular localization pattern; however, this motif is probably altered, or overwhelmed by a new basolateral signal, in the ZIP5 protein.
Sorting signals can be identified via their capability of conferring the trafficking pattern to other proteins (chimeric protein method). In convergence, mutation or deletion of these sorting signals
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can alter the protein’s trafficking pattern (mutational method). These two approaches favor the detection of signals that are conformationally independent or whose conformation is very robust, whereas conformational-sensitive sorting signals are unlikely to be discovered (Vogel et al., 2005).
Because ZIP proteins have similar topology, it is an ideal family to use the chimeric protein method and the mutational method to identify the sorting signal(s) in these proteins.
When we are designing mutational and chimeric protein studies, special attention should be paid to sequence motifs that have previously been demonstrated to play some role in subcellular distribution of the proteins in polarized cells. Unfortunately, despite a lot of effort to identify the sorting signals for protein localization in polarized cells, we still know very little in this field.
Basolateral sorting signals on transmembrane proteins are relatively uniform peptide motifs located on the cytoplasmic tail, generally including a critical tyrosine residue or a dileucine motif, often in the vicinity of patches of acidic amino acids or other uncharacterized motifs (Muth and Caplan,
2003;Rodriguez-Boulan et al., 2005). Recently, a signature sequence QQPFLS in the C-terminus of the NBC1 protein was found to be essential for its exclusive targeting to the basolateral membrane
(Li et al., 2004). In contrast, apical sorting signals are not very homogeneous. Known apical sorting signals include those in glycosylphosphatidylinositol (GPI), N-glycans and O-glycans, and some proteinaceous motifs in the extracellular, transmembrane or cytoplasmic domain (Rodriguez-Boulan et al., 2005).
Summary
Cd is an extremely widespread toxic metal contaminant. Although numerous studies have been carried out over the years, the molecular mechanisms for Cd toxicity were not clear until very recently. With the identification of Slc39a8 as the gene responsible for Cd-induced testicular
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necrosis in inbred mouse strains, a causal effect of a single gene and an in vivo phenotype of Cd toxicity were unequivocally demonstrated. The transport properties of the ZIP8 protein were characterized in detail in a cell culture system, and these properties can be very useful in understanding Cd toxicity at the whole animal level. Besides testis, the ZIP8 transporter is likely to mediate the Cd-toxicity in other target organs of Cd toxicity, such as kidney, lung and liver. Current work in this lab on ZIP8 transgenic mice and ZIP8 knockout mice should enable us to thoroughly assess the in vivo role of ZIP8 in mediating Cd toxicity in different organs and with different exposure routes.
The ZIP8 transporter belongs to the very ancient SLC39 protein family, and there are 14 ZIP members in the human and mouse genomes. Interestingly, ZIP8’s high affinity to Cd was also found for the ZIP14 transporter –the ZIP protein that is most closely related to ZIP8. Thus, the possible role of every ZIP transporter in mediating Cd-toxicity needs to be examined.
Scientific research is a continuous process. The successful answering of one question almost always raises many other (perhaps more interesting) questions. What is the causal mutation for the resistance phenotype of CITN? What is the response element and (or) locus-control element that determines the highly specific expression of ZIP8 in testicular endothelial cells? What is the role of
ZIP8 in maintaining Mn homeostasis (especially in the testis, maybe in the brain as well)? Are
ZIP8 and ZIP14 also important for Zn uptake in any tissues? What is the sequence motif that determines the apical versus the basolateral localization pattern of ZIP proteins?
To answer these questions, the dedication and collaboration of many researchers in this field are needed. And, answering these questions would definitely enrich our knowledge, as well as make us appreciate more of Mother Nature’s beauty.
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Figure Legends
Figure 1 – Analysis of Celera SNPs in Four Mouse Inbred Strains
All SNPs within the 170-kb region defined by the BAC transgene, displayed in the relative 5’ to
3’ order on the BAC. SNP149 to SNP208 (in gray) are located within the Slc39a8 gene
transcript region. SNPs that are identical only for D2 and 129X1 are colored in red, and those
identical only for B6 and A/J are in blue; one of these is highly likely to be the reason for the
genetic difference in expression of ZIP8 in the endothelial cells of the testicular vasculature.
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Figure 1.
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Figure 1.
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