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Characterization of Zinc Transporters in Caco-2 Cells


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CHARACTERIZATION OF ZINC TRANSPORTERS IN CACO-2 CELLS By JEFFREY ALAN BOBO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Jeffrey Alan Bobo

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ACKNOWLEDGMENTS First and foremost, I would like to thank my major professor, Dr. Robert J. Cousins for the opportunity to work in one of the countries top research laboratories. I have grown both as a scientist and a person and I am certain that the knowledge that I have gained from this experience will assist me throughout my lifetime. I would also like to thank my other committee members for their contributions and support towards my project. I would also like to thank my lab mates for their advice, expertise and companionship: Ray Blanchard, Juan Liuzzi, Cal Green, Louis Lichten, Scott Rothbart, Jennifer Moore, Jay Cao, Steve Davis, Ken Chiang, and Khanh Nguyen. I would also like to extent a special thank you to Virginia Mauldin for her kindness, support and words of motivation. Last but not least, I would like to thank my wife, Dr. Rebecca Bobo for her love, support and motivation, despite facing one of the toughest academic challenges herself. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iii LIST OF TABLES ...............................................................................................................v LIST OF FIGURES ...........................................................................................................vi ABSTRACT .....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................2 3 MATERIALS AND METHODS.................................................................................7 Cell Culture...................................................................................................................7 Experimental Design....................................................................................................7 RNA Isolation...............................................................................................................8 Quantitative RT-PCR....................................................................................................8 Protein Isolation............................................................................................................9 Antibody Production.....................................................................................................9 Western Analysis........................................................................................................10 Electron Microscopy...................................................................................................10 Statistics......................................................................................................................11 4 RESULTS...................................................................................................................13 RNA Expression.........................................................................................................13 Protein.........................................................................................................................14 Electron Microscopy...................................................................................................15 5 DISCUSSION.............................................................................................................30 LIST OF REFERENCES...................................................................................................34 BIOGRAPHICAL SKETCH.............................................................................................38 iv

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LIST OF TABLES Table page 3-1 Peptides used to generate polyclonal antibodies to ZnT transporter proteins..........12 3-2 Peptides used to generate polyclonal antibodies to Zip transporter proteins...........12 v

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LIST OF FIGURES Figure page 4-1 ZnT1 zinc supplementation time course..................................................................16 4-2 ZnT1 TPEN treatment time course..........................................................................16 4-3 ZnT4 zinc supplementation time course..................................................................17 4-4 ZnT4 TPEN treatment time course..........................................................................17 4-5 ZnT5 zinc supplementation time course..................................................................18 4-6 ZnT5 TPEN treatment time course..........................................................................18 4-7 ZnT6 zinc supplementation time course..................................................................19 4-8 ZnT6 TPEN treatment time course..........................................................................19 4-9 ZnT7 zinc supplementation time course..................................................................20 4-10 ZnT7 TPEN treatment time course..........................................................................20 4-11 Zip1 zinc supplementation time course....................................................................21 4-12 Zip1 TPEN treatment time course............................................................................21 4-13 Zip3 zinc supplementation time course....................................................................22 4-14 Zip3 TPEN treatment time course............................................................................22 4-15 Zip4 zinc supplementation time course....................................................................23 4-16 Zip4 TPEN treatment time course............................................................................23 4-17 Zip1 mRNA expression in Caco-2 cells after 72 hr treatment with 50 ng/ml ILl ..........................................................................................................................24 4-18 ZnT2 expression comparing vehicle control (100% EtOH) with treatment of 10 ng/ml calcitriol after 72 hrs......................................................................................24 4-19 Zip1 dot blot ...........................................................................................................25 vi

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4-20 Comparison Zip1 protein expression after 24 h treatment of 5.0 M TPEN or 80 M zinc..............................................................................................................25 4-21 Effect of 50 ng/ml of IL1 on Zip1 protein after treatment for 24 h.......................26 4-22 Effect of 10 ng/ml of calcitriol on ZnT2 protein expression....................................27 4-23 Electron micrograph of apical side of Caco-2 cell cells...........................................28 4-24 Electron micrograph of control Caco-2 cells...........................................................28 4-25 Electron micrograph of IL1 treated Caco-2 cells..................................................29 vii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CHARACTERIZATION OF ZINC TRANSPORTERS IN CACO-2 CELLS By Jeffrey Alan Bobo August 2004 Chair: Robert J. Cousins Major Department: Food Science and Human Nutrition Zinc is an essential micronutrient that is tightly regulated in humans and other animals and requires specialized mechanisms for its uptake, transport and excretion. Zinc metabolism has been extensively studied in both man and rodents, and zinc transport has received much attention with in vitro models. Numerous investigators have used isolated cell types, including hepatocytes, fibroblasts, endothelial cells and intestinal cells to help further characterize zinc transport. Of particular interest is the intestinal Caco-2 cell model. Caco-2 cells, a human colonic tumor cell line, have been studied considerably in the last decade as a model for drug, macronutrient and micronutrient absorption and transport. There are several characteristics that render Caco-2 cells an ideal model for absorption and transport. These cells can grow in monolayers and, upon confluency, the cells differentiate and become polarized. They form tight junctions and possess a brush border with functional brush border enzymes and thus resemble enterocytes. Due to these inherent characteristics, we have focused on Caco-2 cells to characterize the regulation of expression of the two zinc transporter gene families, ZnT viii

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and Zip, under conditions of zinc deprivation and supplementation. The current hypothesis is that ZnT family transporters decrease intracellular zinc concentration, while Zip family transporters increase intracellular zinc concentration. The human zinc transporter genes studied were ZnT1, 4, 5, 6, and 7, and Zip-1, -3, and -4. The Caco-2 cells were treated with TPEN, a cell permeable zinc chelator, at 1.25, 2.5 or 5.0 M, zinc sulfate at 40 or 80 M, IL1 or calcitriol. Transporter expression was measured by quantitative real-time PCR, western blotting and immunohistochemistry. The results from this study demonstrate that some transporters from both families show differential regulation of expression based on treatment, while others were refractory to change due to zinc status. ZnT1, 4, 7 and Zip4 expression was decreased by zinc deprivation, while ZnT-6 was up-regulated. Zinc restriction up-regulated Zip1, while Zip3 was down-regulated. Zip1 was upregulated by IL1 while ZnT2 was downregulated by calcitriol. Overall, the results suggest genes from both zinc transporter families are differentially expressed in Caco-2 cells in a mode consistent with maintenance of cellular zinc homeostasis in zinc restriction and supplementation, and certain zinc transporter genes are affected by physiological stimuli. ix

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CHAPTER 1 INTRODUCTION Zinc is intimately involved in many metabolic processes in the body including DNA replication, immune function and protein-protein interaction. Subsequently is it an essential micronutrient that is regulated under tight control in humans and other animals and requires specialized mechanisms for transport (1, 2). There are a number of zinc transporters that are involved in the transport and metabolism of zinc either through efflux or influx (3, 4) and these transporters are from two distinct gene families, ZnTs and ZiPs. Over the last several years numerous studies have been conducted and there have been several discoveries of additional transporters in both of these families. The purpose of this study is to characterize the various zinc transporters from the ZnT and ZiP families found in Caco-2 cells and to further understand the mechanisms by which zinc is absorbed and transported in Caco-2 cells. 1

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CHAPTER 2 LITERATURE REVIEW Zinc is an essential divalent cation that is required for many biochemical processes in mammals. It is a strong Lewis acid and does not demonstrate any redox chemistry, which is why it is involved in many biological processes (1). The biochemical roles of zinc can be divided into three different categories, catalytic, structural and regulatory. Catalytically zinc has a concentrated 2 + charge, which results in high electron affinity and makes it a good attacking group. There are over 300 known zinc metalloenzymes, which require zinc for function, some of which are involved in gene transcription, i.e. RNA polymerases. The structural role of zinc is also a result of its chemistry and is demonstrated through zinc finger proteins. Zinc fingers are regions in proteins that have a structure of cysteine and histidines to achieve a unique structure that allows the binding of zinc. These proteins have a variety of functions that include transcription factors, RNA binding proteins and protein-protein interactions. The regulatory role of zinc is seen in the regulation of metal response elements, or MREs. MREs are located in the promoter regions of regulated genes and can interact with metal-binding transcription factors, MTFs. MTFs are zinc finger proteins that acquire zinc from the cytosol, translocate to the nucleus and interact with MREs to stimulate transcription (1). Due to the wide variety of essential roles mentioned above, it is easy to see why homeostatic control of zinc levels is important. Even a minor zinc deficiency could potentially cause a decrease in one of the functions of this micronutrient that could influence health. Clinical manifestations due to mild to severe zinc deficiency range 2

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3 from alopecia, acrodermatitis, diarrhea, impaired wound healing and immunity, as well as growth retardation, and hypogonadism. Therefore it is important to understand the absorption of this essential micronutrient from the gastrointestinal tract. Zinc is absorbed throughout the small intestine, but studies suggest that the duodenum and the jejunum are the primary sites (5, 6, 7). In order to maintain zinc homeostasis, a balance must exist between absorption of dietary zinc and endogenous secretions through adaptive regulation to dietary zinc (8). The intestine is the key organ in maintaining zinc balance through absorption and endogenous excretion. It has been demonstrated in humans and animals that a restriction of dietary zinc results in increased uptake and efficiency of intestinal absorption (9-11). Zinc transport has received much attention through research with in vitro models. Numerous investigators have used isolated cell types, including hepatocytes (12-14), placental trophoblasts (15), fibroblasts (16), intestinal cells (17), endothelial cells (18), and membrane vesicles (19-22) to help further characterize zinc transport. Studies using radioisotopes have identified and given insight into zinc processing and pathways that result in the overall homeostatic control of zinc levels in the body (3). Though the models differ, there are several distinct characteristics that can be concluded. Zinc transport is a time-, concentration-, pH-, and temperature-dependent process, with some evidence demonstrating saturable and nonsaturable components (2). Metabolic studies have shown that dietary zinc intake influences intestinal absorption, while certain hormones, cytokines and growth factors influence zinc metabolism in specific certain organ systems (23-25).

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4 Since the intestine is the key regulator of zinc homeostasis, intestinal cell models are of particular interest. Caco-2 cells, a human colonic tumor cell line, have been studied considerably in the last decade as a model for drug, macronutrient, and micronutrient absorption and transport. There are several characteristics that render Caco-2 cells an ideal model for absorption and transport. These cells grow in monolayers and upon confluency, the cells differentiate and become polarized. They form tight junctions and possess a brush border with functional brush border enzymes and thus, resemble enterocytes (17). Transport and absorption has been shown to be regulated in Caco-2 cells for several nutrients including iron, glucose, calcium and zinc. Treatment of Caco-2 cells with high concentrations of iron caused DMT1, an iron transporter protein typically located at the apical membrane to decrease while total cellular DMT1 levels remained steady (26). These results demonstrate that in order to decrease iron absorption the transporter protein is able to translocate based on the needs of the cell. It has also been shown that different isoforms of the glucose transporters (GLUT) are differentially localized (27). Fleet and Wood (28) used Caco-2 cells to investigate the effects of active vitamin D on calcium transport. They showed that calcium transport increases upon addition of calcitriol to the medium. Focusing on zinc transport, Raffaniello et al. (17) demonstrated there were two distinct pathways at the apical and basolateral sides in Caco-2 cells as well as a vesicularly mediated component. Additionally, Fleet et al. (29) noticed that Caco-2 cells treated with calcitriol increased zinc transport by 159% and that this increase was not due to lysosome-mediated zinc transport. The molecular mechanisms of zinc transport and

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5 absorption are not fully known but involve brush border transport, intracellular transport and basolateral transport (30). In the last decade, there have been numerous studies that have provided evidence that there are a number of transporter proteins that are involved in zinc transport in mammals. The first zinc transporter gene cloned was ZnT1 by Palmiter and Findley (31). Since its discovery, several other zinc transporters have been discovered and characterized. In fact, there are actually two distinct gene families of zinc transporters, ZnTs and Zips (3). The current hypothesis is that ZnT proteins reduce intracellular zinc either by efflux from the cell or by sequestering zinc within vesicles. The ZiP proteins are thought to increase intracellular zinc through extracellular uptake or by transport from vesicles into cytoplasm (3). Brush border uptake characteristics by enterocytes suggest both saturable and nonsaturable components at low and high luminal zinc concentrations, respectively (30). The transporters thought to be located apically, and thus responsible for luminal uptake or cellular efflux are Zip1, 2, 4 and ZnT5 (3). Interestingly, it was found by Wang et al. (32) that a mutation in the gene that encodes for Zip4 is the cause for the disease acrodermatitis enteropathica, in which patients suffer from a severe zinc deficiency due to malabsorption of dietary zinc. It is known that protein synthesis is required for the downregulation of zinc absorption under high concentrations of zinc (33) and that metallothionien (MT) is a major zinc binding protein in intestinal cells. MT has been used as the sentinel gene for cellular zinc status. Its expression is increased by high dietary zinc and decreased with lower zinc intake. The current hypothesis is that MT acts as a buffer for intracellular zinc

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6 (34) so it controls how much zinc can be transferred to the basolateral and apical surfaces for either transport into circulation or back into the lumen, respectively. Zinc absorption decreases as MT synthesis rises in response to dietary zinc. As mentioned above, there is also evidence of a vesicularly or lysosomal mediated component of zinc transport in Caco-2 cells (17, 29). At this time the function of this phenomenon is not known, however, zinc transport in to and out of the vesicles could be a component of the absorption process. The transporters that are thought to be located vesicularly are ZnT2, 4, 6, and 7 and Zip1 and 3. Evidence suggests that basolateral zinc transport is comprised of both saturable and nonsaturable components in both directions (34, 35). Basolateral transfer appears to be linear and zinc concentration dependent. Endogenous zinc returns to the intestine as enterocytes slough off into the lumen or as pancreatic secretions. The zinc transporters that are thought to be involved in zinc trafficking by enterocytes have not been identified. The purpose of the current study is to gain insight into the regulation of zinc homeostasis by the intestine using Caco-2 cells as a model in order to see the changes in expression of the zinc transporter genes under conditions of zinc deprivation, zinc supplementation and other physiological factors, including IL1 and calcitriol.

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CHAPTER 3 MATERIALS AND METHODS Cell Culture Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD). They were maintained in DMEM under standard conditions: 37 C and 5% CO 2 Cells were in medium containing 10% v/v FBS. Once experimental design was planned, cells were seeded into the desired number of T-25 culture flasks at approximately 3.5 x 10 5 cells/cm 2 The following day the meduim was changed to DMEM with 5% v/v FBS and MITO+ cell differentiation/ serum extender supplement (BD Biosciences) in order to induce differentiation of the Caco-2 cells. The cells were then allowed to differentiate for five to seven days with medium changes every 48 h prior to experimentation. Experimental Design Cells were treated in order to mimic conditions of either a zinc deprived state or zinc excess. Zinc deprivation was produced using (TPEN), a cell permeable zinc chelator with a high affinity for zinc (K d = 10 -16 M). The medium with 5% v/v FBS has a zinc concentration of approximately 1.54 M. Since TPEN has a 1:1 binding capacity with zinc, three different TPEN concentrations were used: 1.25, 2.5 and 5.0 M, in order to mimic a moderate to severe zinc deficiency. Zinc sulfate was used to supplement the medium with excess zinc (40 and 80 M). For both treatment conditions, TPEN and excess zinc, a time course was performed in order to discover the largest change in transporter transcript expression. The time points used were 0, 4, 8, 12, 24, and 48 h. 7

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8 Cells were also treated with IL1B, a proinflamatory cytokine, at 50 ng/ml or calcitriol, the hormonal form of vitamin D, at 10 ng/ml for 24 h. RNA Isolation For each T-25 culture flask, one ml of TriPure Isolation Reagent (Roche Molecular Biochemicals, Indianapolis, IN) was added after removal of the medium. The cells were then scraped from the flask and vigorously triturated for 1 min. The sample was then put into microfuge tubes and stored at o C until all RNA was harvested for the entire time course. Samples were then thawed, 0.2 ml of chloroform was added, followed by vigorous shaking for 15 sec. The samples were then centrifuged for 10 min at 4 o C at 12,000 x g and the clear aqueous layer was transferred to a new sterile tube and 0.5 ml of isopropyl alcohol was added to precipitate RNA. Samples were gently mixed and left sitting at room temperature for 10 min and then centrifuged again for 10 min at 12,000 x g. RNA pellets were then washed 3 times with 75% v/v ethanol followed by centrifugation at 7500 x g for 5 min. Following ethanol washes, RNA pellets were resuspended in 200 all of diethyl carbonate (DEPC)-treated water. Purity and concentration were determined spectrophotometrically as described by Moore et al. (36). Each sample was then aliquoted (50 l) and treated with DNase I (Ambion, Austin, TX) to eliminate DNA contamination. Quantitative RT-PCR Primers and probes for each transporter gene were designed using Primer Express software (Applied Biosystems, Foster City, CA) based on sequence information for each gene. The relative quantity of RNA was determined by using 18S rRNA (primer and probes designed by Applied Biosystems) for normalization. A standard curve was generated using 1:10 dilutions of total RNA starting at 10ng/ml and producing a 4to 5

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9 log range for each determination. The ABI Model 5700 Sequence Detection System was used to fluorometrically measure amplification of PCR products for each transporter gene transcript. Protein Isolation Cells were grown and treated as described above except T-75 culture flasks were used. The standard treatment time was 24 h. Cells were rinsed twice with PBS prior to the addition of 5 ml of a modified RIPA buffer containing: 1%Triton x-100, 0.5% w/v sodium deoxycholate, 1% v/v SDS in PBS with protease inhibitor cocktail P-2714 (Sigma, St. Louis, MO). Cells were scraped from the flasks and then triturated with a 25 gauge needle in order to shear the nucleic acids in the sample. The protein concentration of the total cell preparation was then determined by colorimetric assay (37). A standard curve was established using bovine serum albumin and the absorbance was read at 650 nm. Linear regression was then used to determine protein concentration. Antibody Production Peptides (Biosynthesis Incorporated, Lewisville, TX), shown in Table 1 and 2, were coupled to KLH (Pierce, Rockford, IL) and used to generate polyclonal antibodies, as described previously (38). Total IgG was isolated by the precipitation method (39). Caprylic acid was added to the serum (pH 5.0 with acetic acid) in order to precipitate serum proteins other than IgGs. Serum was centrifuged for 30 min at 35,000 x g. Supernatant was removed and ammonium sulfate was added (1:1, v/v) to precipitate the total IgG fraction. Some of the total IgG fractions were then affinity purified (AP) (Sulfolink; Pierce). Specificity of the affinity purified antibodies and IgG fractions were determined by comparing signals from pre-immune serum, total IgG and where applicable, affinity purified IgG and flowthrough effluent from the affinity

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10 chromatography. The methods are comparable to those reported by McMahon and Cousins (4). Western Analysis Equal amounts (30 g) of total cellular protein were resolved by 10% SDS-PAGE gels and transferred to PVDF (Immobilon; Millipore, Bedford, MA) in standard Towbin transfer buffer (20% w/v methanol). Blots were stained with Ponceau Red to verify equal protein loading and then destained. Membranes were wetted with methanol and then placed in Milli-Q water to allow complete wetting, followed by blocking in 5% w/v nonfat dry milk (Carnation) in PBS-T (0.025% v/v Tween 20). Membranes were washed for 5 min in PBS-T and then incubated for 1 h in PBS-T with 1:100 of whole serum, 10-20 g/ml of total IgG or 2-4 g/ml of affinity purified IgG; followed by three 5 min washes with PBS-T. Membranes were incubated with anti-rabbit IgG secondary antibody conjugated to alkaline phosphatase (Amersham Biosciences, Piscataway, NJ) for 1h, washed 3 x for 5 min each and then viewed with a chemiluminescent reagent for detection (Amersham Biosciences) using fluorescence imaging (Molecular Dynamics Storm 840 Imager). Electron Microscopy Caco-2 cell monolayers were grown and allowed to differentiate as previously mentioned on fibrillar collagen I inserts (Becton Dickinson Labware, Bedford, MA). Cells were rinsed 2 x with sterile PBS and fixed in a 0.1 M sodium cacodylate buffer containing 4 % w/v paraformaldehyde and 0.5 % w/v glutaraldehyde for 24 hrs at 4 o C at which time the fixative was changed to a 0.1 M cacodylate buffer. Cells were stored in this buffer at 4 o C until further processing.

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11 Inserts with attached cells were then dehydrated through a series of ethanol washes for 15 min each. The first wash was 15% v/v ethanol followed by 20%, 25%, 30%, 50%, 70%, and 90%. The next incubation was in 90% ethanol mixed 1:1 with the LR White (Electron Microscope Sciences, Hatfield, PA) embedding medium for 10 min, followed by a 10 min incubation in 100% LR White. The membranes were then excised from the insert chamber and then cut in half and once again incubated in 100% LR White for 20 min, then one h and then left rotating overnight at room temperature. The next morning each membrane was placed into fresh LR White and then kept in a 60 o C oven overnight in gelatin capsules to harden. Extra embedding material was trimmed away in order to cut sections perpendicular to the plane of the membrane. Sections were cut 80-90 nm thick and mounted on formvar nickel coated 200 mesh grids (Electron Microscope Sciences, Hatfield, PA). Grids were blocked in 1% w/v BSA in sterile filtered PBS for 15 min and rinsed in PBS-T (0.025% v/v Tween 20) for 1 min. Grids were incubated in PBS-T containing primary antibody diluted about 1:100 and placed in a dark, damp chamber for 1 h. Grids were washed 3 x in PBS-T for 3 min each followed by 2 washes in PBS for 3 min. Samples were then placed in secondary, immunogold labeled (18 nm) antibody solution (1:50) (Jackson Immunoresearch, West Grove, PA) and incubated for 1 h, followed by 3, 5 min washes in PBS, a 5 min incubation with Trumps and 4, 5 min washed in Milli Q Water and one 15 s jet wash. Specimens were viewed with a Hitachi H-7000 Transmission Electron Microscope (ICBR EM Core Laboratory, University of Florida). Statistics Each data point represented throughout the project was a replicate of at least three different trials (n=3), unless otherwise noted. A one-way ANOVA was conducted in

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12 order to determine if there were any differences between data groups. The post hoc test was performed using the Student Newman-Keuls multiple comparison test. Differences were considered significant with a p-value of equal to or less than 0.05. Table 3-1.Peptides used to generate polyclonal antibodies to ZnT transporter proteins. Transpor ter e 1 KE 2 SHP T4 WEE NVLNFSDHHVIP/h/rZnT8 KPVNKDQCP able 3-2. Peptides used to generate polyclonal antibodies to Zip transporter proteins. NHEASASG m/rZIP2 EEWGGTHAFGFH /rZIP3 RLRELGRPG EETPELLNPETRRL Peptid m/rZnT GTRPQVHSG m/rZnT CHAQKDSG m/r/h/Zn MQLIPGSSSK m/hZnT5 mZnT6 SPSKRGQKGTLI VAP m Q T Transporter Peptide m/hZIP1 RSGA E m CAGL mZIP4 A

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CHAPTER 4 RESULTS RNA Expression Zinc transporter transcripts that were detected in Caco-2 cells using quantitative PCR (Q-PCR) were ZnT1, 4, 5, 6, and 7 and Zip1, 3, and 4. Transcript data at each time point are presented on a relative basis where vehicle control expression levels are equal to one. ZnT1 expression increased 2-fold by 4 h in response to supplemental zinc (Zn+) (Fig 4-1). Levels remained elevated for 24 h and then returned to basal conditions. ZnT1 mRNA levels were greater at 80 M Zn than 40 M Zn. Figure 4-2 shows that ZnT1 expression decreased rapidly with all concentrations of TPEN. The response to zinc deprivation (Zn-) with TPEN chelation was greatest after 4 h. Figures 4-3 illustrates that ZnT4 decreased steadily starting at 12 h with 80 M zinc treatment reaching 2-fold at 48 h, while ZnT4 mRNA levels decreased only in the more severe Znconditions, reaching a decrease of nearly an 8-fold after 24 h of treatment with 5.0 M TPEN (Fig 4-4). ZnT-5 transcript levels were refractory to any significant changes for either Zn+ and Znthrough out the time courses except a 9-fold decrease at 48 h with 5.0 M TPEN (Figs 4-5 & 4-6). However, the 48 h time point with 5.0 M TPEN treatment was not considered valid because cells were more than 50% apoptotic by trypan blue exclusion (data not shown). ZnT6 transcript expression was refractory to change under zinc supplementation (Fig 4-7), but treatment 5.0 M TPEN treatment increased ZnT6 expression up to 2-fold by 12 h (Fig 4-8). Conditions of zinc excess had no effect on ZnT7 expression levels (Fig 4-9). In contrast, conditions of mild zinc deprivation (1.25 M TPEN) had no effect 13

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14 on ZnT7 expression, while 2.5 M TPEN increased expression 2-fold at 12 h and 48 h with 2.5 M TPEN and 5.0 M TPEN decreased transcript levels 3-fold after 24 h (Fig 4-10). Zinc supplementation caused a 2-fold decrease in expression of the Zip1 transcript at 12 h (Fig 4-11). TPEN treatment of 2.5 and 5.0 M caused over a 2-fold increase in Zip1 expression starting at 12 h and continuing through 24 h (Fig 4-12). Zip3 transcript expression was not significantly altered by zinc excess, however zinc deprivation caused a steady decrease in expression starting at 8 h and resulting in an 8-fold decrease by 24 h (Figs 4-13 & 4-14). Zip4 also had no significant changes in transcript expression with zinc supplementation, while zinc deprivation decreased Zip4 expression almost 2-fold (Fig 4-15 & 4-16). Two of the zinc transporters, one from each gene family, Zip1 and ZnT2, were responsive to the two physiological stimuli tested. Zip1 was the only transporter that was regulated by IL1, increasing RNA expression almost 5-fold over the controls as seen in Figure 4-17. ZnT2 was the only transporter that changed mRNA levels when cells were first treated with 10 nmol calcitriol for 72 h compared to control cells. Expression decreased approximately 3-fold when compared to control cells (Fig 4-18). Protein Preimmune serum (PIS), affinity purified IgG (AP), and the flow through (FT, affinity chromatography effluent) were used to determine the specificity of the antibodies utilized for further examination of Zip1 and ZnT2 transporters (Fig 4-19). Protein expression for Zip1 comparing Zn+ to Znshows that zinc deprivation increased Zip1 expression while zinc excess decreased protein expression by the 24 h time point (Fig 4

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15 20). In addition, treatment with IL1 caused a 3.5-fold increase in Zip1 protein expression. ZnT2 protein expression was decreased nearly 2-fold with 10 nmol/ml of calcitriol by the 24 h time point (Figure 4-21). Electron Microscopy Electron micrographs (Fig 4-23 & 4-24) illustrate that Zip1 is located vesicularly throughout cytoplasm and to a smaller extent to the brush border membrane. Figure 4-25 demonstrates that upon treatment with IL1 Zip1 in upregulated, which is in agreement with mRNA and western data.

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16 ZnT-1 mRNA Expression in Caco-2 Cells: Zn Supplementation Time Course0.111048122448Time (hrs)mRNA Expression Realative to Control 40 uM 80 uM ZnSO 4 Figure 4-1. ZnT1 zinc supplementation time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3). ZnT-1 TPEN Time Course0.010.111048122448HoursRelative mRNA Levels [1.25] [2.5] [5.0] TPEN (uM) Figure 4-2. ZnT1 TPEN treatment time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3).

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17 ZnT-4 mRNA Expression in Caco-2 Cells: Zn Supplementation Time Course0.111048122448Time (hrs)mRNA Expression Realative to [0] Control 40 uM 80 uM ZnSO 4 Figure 4-3. ZnT4 zinc supplementation time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3). ZnT-4 TPEN Time Course0.010.111048122448HoursRelative mRNA Levels [1.25] [2.5] [5.0] TPEN (uM) Figure 4-4. ZnT4 TPEN treatment time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3).

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18 ZnT-5 mRNA Expression in Caco-2 Cells: Zn Supplementation Time Course0.111048122448Time (hrs)mRNA Expression Realative to Control 40 uM 80 uM ZnSO 4 Figure 4-5. ZnT5 zinc supplementation time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3). ZnT-5 TPEN Time Course0.111048122448HoursRelative mRNA Levels [1.25] [2.5] [5.0] TPEN (uM) Figure 4-6. ZnT5 TPEN treatment time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3).

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19 ZnT-6 mRNA Expression in Caco-2 Cells: Zn Supplementation Time Course0.111048122448Time (hrs)mRNA Expression Realative to Control 40 uM 80 uM ZnSO 4 Figure 4-7. ZnT6 zinc supplementation time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3). ZnT-6 TPEN Time Course0.111048122448HoursRelative mRNA Levels [1.25] [2.5] [5.0] TPEN (uM) Figure 4-8. ZnT6 TPEN treatment time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3).

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20 ZnT-7 mRNA Expression in Caco-2 Cells: Zn Supplementation Time Course0.111048122448Time (hrs)mRNA Expression Realative to Control 40 uM 80 uM ZnSO 4 Figure 4-9. ZnT7 zinc supplementation time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3). ZnT-7 TPEN Time Course0.111048122448HoursRelative mRNA Levels [1.25] [2.5] [5.0] TPEN (uM) Figure 4-10. ZnT7 TPEN treatment time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3).

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21 ZiP-1 mRNA Expression in Caco-2 Cells: Zn Supplementation Time Course0.111048122448Time (hrs)mRNA Expression Realative to [0] Control 40 uM 80 uM ZnSO 4 Figure 4-11. Zip1 zinc supplementation time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3). ZiP-1 TPEN Time Course0.111048122448HoursRelative mRNA Levels [1.25] [2.5] [5.0] TPEN (uM) Figure 4-12. Zip1 TPEN treatment time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3).

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22 ZiP-3 mRNA Expression in Caco-2 Cells: Zn Supplementation Time Course0.111048122448Time (hrs)mRNA Abundance Realative to [0] Control 40 uM 80 uM ZnSO 4 Figure 4-13. Zip3 zinc supplementation time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3). ZiP-3 TPEN Time Course0.111048122448HoursRelative mRNA Levels [1.25] [2.5] [5.0] TPEN (uM) Figure 4-14. Zip3 TPEN treatment time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3).

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23 ZiP-4 mRNA Expression in Caco-2 Cells: Zn Supplementation Time Course0.111048122448Time (hrs)mRNA Expression Realative to Control 40 uM 80 uM ZnSO 4 Figure 4-15. Zip4 zinc supplementation time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3). ZiP-4 TPEN Time Course0.111048122448HoursRelative mRNA Levels [1.25] [2.5] [5.0] TPEN (uM) Figure 4-16. Zip4 TPEN treatment time course. Values at each time point are relative to vehicle control (vehicle control=1 at each time point). Data represented by mean standard deviation (n=3).

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24 Caco 2 Cells hZiP-1 mRNA IL-B Treatment01234567Fold Change hZiP-1 mRN A Control IL-1 B Figure 4-17. Zip1 mRNA expression in Caco-2 cells after 72 hr treatment with 50 ng/ml ILl ZnT2 mRNA Expression after 72 hr Treatment with Calcitriol00.20.40.60.811.2ControlCalcitriolRelative abundance of mRNA Figure 4-18. ZnT2 expression comparing vehicle control (100% EtOH) with treatment of 10 ng/ml calcitriol after 72 hrs. Note: These data (calcitriol mRNA experiment only) reflect a pilot experiment that was conducted (n=1). Further testing would be appropriate to confirm these results)

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25 A. B. C. D. Figure 4-19. Zip1 dot blot: A. Immune serum and B. Pre-immune serum, C & D. Western blot: C. Affinity purified antibody and D. Flow through. ZiP-1 Protein in Caco-2 Cells:00.511.522.535uM TPEN80uM ZnTreatmentRelative Densotometric Units Figure 4-20. Comparison Zip1 protein expression after 24 h treatment of 5.0 M TPEN or 80 M zinc (n=3).

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26 Zip1 Protein IL1 B Exp.00.511.522.533.544.55ControlIL1 BRelative Densitometric units Figure 4-21. Effect of 50 ng/ml of IL1 on Zip1 protein after treatment for 24 h (n=3).

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27 ZnT2 Protein: Calcitriol treatment00.20.40.60.811.21.4ControlCalcitriolRelative Densotometric Units Figure 4-22. Effect of 10 ng/ml of calcitriol on ZnT2 protein expression (n=3).

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28 A. B.. C.. D. Figure 4-23. Electron micrograph of apical side of Caco-2 cell cells. A. and B. are negative controls. (No primary antibody was used, but all other immunolabeling steps were conducted). C. and D. Represent control cells that were immunogold-labeled with polyclonal Zip1 antibody along the apical membrane. The arrows are pointing to 18 nm gold particles attached to secondary antibody. A.. B.. Figure 4-24. Electron micrograph of control Caco-2 cells. A. Shows positive labeling between nucleus (left side) and apical membrane. B. Shows some positive labeling just outside the nucleus, possibly representing the rough endoplasmic reticulum.

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29 A. B. D. C. Figure 4-25. Electron micrograph of IL1 treated Caco-2 cells. A. and B. Show considerable labeling throughout the cytoplasm and along the brushborder. C. Represents labeling around a junction between two cells. D. demonstrates labeling around the nucleus of two cells.

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CHAPTER 5 DISCUSSION The results from this project demonstrate that there is differential regulation of expression of the zinc transporters from both gene families, Zip and ZnT, in Caco-2 cells. These data give us further insight into the mechanistic control of zinc absorption, however one must consider that this is an in vitro model using a transformed cell line. ZnT1 expression was consistent with current literature as far as its response to low and high zinc levels in both cell culture and animal studies (4). ZnT1 had a quick response (within 4 hrs) to both zinc deprivation and supplementation. Interestingly, the lowest TPEN treatment (1.25 M), used to mimic a mild deficiency, decreased ZnT1 expression by 2-fold. These data indicate that ZnT1 regulated in a way that allows it to rapidly adapt to environmental conditions and may be part of the first defense against environmental changes in the zinc supply. Furthermore, these data support that ZnT1 functions as a cellular zinc exporter which downregulates to conserve zinc. In addition, Palmiter (40) has shown there to be an interaction between ZnT1 and metallothionein, the sentinel gene for cellular zinc status, which further suggests ZnT1 has a role in adaptation to differential zinc concentrations within cells. ZnT4 was essentially refractory to zinc supplementation. Similarly, only severe zinc depletion resulted in ZnT4 downregulation. Assuming that ZnT4 is vesicular and exports zinc from the cytoplasm into vesicles, that the two lower TPEN treatments produce ZnT4 is downregulation indicates that this transporter may conserve zinc that is 30

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31 left in the cell. Alternatively, the function of ZnT4 may not be critical for survival and begins to shut down during a severe zinc deficiency. There was no change in ZnT5 within the 48 h time period examined which could indicate that ZnT5, shown to be located apically (41) in intestinal cells is refractory to change based on nutrient status. However, Cragg et al. (41) showed that ZnT5 increased after treatment of Caco-2 cells with 100 M zinc for 7 d. Perhaps ZnT5 expression changes only during chronic conditions of zinc excess. ZnT6 significantly increased only at 12 h with 2.5 and 5.0 M TPEN. This could signify that ZnT6 may serve to sequester zinc once the cell detects that levels are dropping in order to conserve zinc for vital function. This hypothesis is in agreement with the observation that there is no change in ZnT6 with zinc supplementation. ZnT7 distribution is in small intestine, liver and spleen and to a lesser extent in the kidney lung and brain. It is located vesicularly and functions to accumulate zinc into the golgi apparatus (42). In this study, zinc supplementation had no effect on ZnT7 expression. Since ZnT7 mRNA upregulates with mild TPEN treatment, our results agree with the conservation (accumulation) concept. However, as shown with ZnT5 and ZnT6, 5.0 M TPEN decreases ZnT7 expression and may demonstrate a level of functional priority necessary for cell survival. The increase in Zip1, which was found localized apically as well as vesicularly, follows the current hypothesis that this transporters function is to increase the intracellular zinc concentration (3). From the data collected, Zip1 is thought to be the major importer of cellular zinc, or at least the importer that is the most regulated in Caco-2 cells. This information somewhat agrees with data that Wang et al. (43) found in

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32 transfected HEK 293 cells showing that under Znconditions, Zip1 localizes to the plasma membrane. The location of Zip3 in intestinal cells is not known, but Wang et al. (43) showed that Zip3 cDNA stimulates zinc uptake when transfected into HEK293 cells and that the subcellular distribution of Zip3 is zinc regulated. They found that Zip3 expression at the plasma membrane increased only after more severe zinc deprivation compared to Zip1. Zip3 mRNA expression was significantly downregulated by severe zinc deprivation in this study, suggesting that its function in the intestine may differ from that in transfected kidney cells. The function of Zip3 in Caco-2 cells will be more clear once its intracellular localization is established. Zip4 is the gene that is thought to be the most important for zinc absorption since it is responsible for the disease Acrodematitis Enteropathica. In this genetic disease patients exhibit zinc malabsorption and suffer from a severe zinc deficiency due to a mutation of the Zip4 gene. The results presented in this project differ with respect to Zip4 expression in mice, where zinc deficiency produces a marked increase in expression (Liuzzi unpublished data). There are several reasons transporter expression may vary among cell types and between cell lines. Caco-2 cells, though a good model, have such limitations. The first and most obvious reason is that these cells are not enterocytes. They are transformed colonic cells that have enterocyte-like properties. Additionally, this study has shown that Zip1 and ZnT2 are responsive to IL1 and vitamin D treatments, respectively. Il1 was used because it was shown to induce IL8 secretion in Caco-2 cells (44) and we were interested to see if there was any change in zinc transporter expression due to an induced

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33 immune response as might be seen in inflammatory bowel disease. Calcitriol was shown to increase zinc transport in Caco-2 cells by 150% by Fleet et al. (29) so we were interested to see which transporters might be responsible for the increased zinc transport. The reduction in ZnT2 expression by calcitriol suggests this hormonal form of vitamin D might stimulate zinc absorption by decreasing intestinal retention. ZnT2 has been related to intestinal zinc storage (38). The fact that physiological and not just nutrient stimuli effect regulation shows that there may be several different signaling systems responsible for regulation of zinc transporter expression in an intact animal. None of the Zip transporters that were found in Caco-2 cells, Zip1, 3, and 4 were found to be highly regulated by zinc supplementation. The data from this project reflect the first model using Caco-2 cells to survey an array of zinc transporters and may serve as a springboard for future research. This current project uses RNA and some protein data to further understand the functions of these transporters based on observed expression changes due to certain external stimuli, nutrient or physiological. It shows that there are several levels of differential expression for each of the transporters. Continued study using this cell model to predict function and roles should lead to a greater understanding of transporter function. The fact that many of these transporters respond directly to a nutrient excess or deprivation lean to the notion that they exhibit zinc responsiveness which is an elementary mechanism of nutrient regulation.

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LIST OF REFERENCES 1. Cousins, R. J. (1986) Toward molecular understanding of zinc metabolism. Clin. Physiol. Biochem. 4: 20-30. 2. Reyes, J. G. (1996) Zinc transport in mammalian cells. Am. J. Physiol. 270: C401-C410. 3. Liuzzi, J. P. and Cousins, R. J. (2004) Mammalian zinc transporters. Annu. Rev. Nutr. 24: 151-172. 4. McMahon, R. and Cousins, R.J. (1998). Regulation of the zinc transporter ZnT-1 by dietary zinc. Proc. Natl. Acad. Sci. USA 95(9):4841-6. 5. Solomons, N.W. and Cousins, R.J. (1984). Zinc. In: Solomons N.W., Rosenburg, I.H. (eds), Absorption and malabsorption of mineral nutrients. Alan R. Liss, New York, pp 125-197. 6. Cousins, R.J. (1989). Theoretical and practical aspects of zinc uptake and absorption. In Laszlo J.A., Dintis, F.R. (eds), Mineral absorption in the monogastric GI tract: chemical, nutritional, and physiological aspects. Plenum, New York, pp 3-12. 7. Lee, H.H., Prasad, A.S., Brewer, G.I. and Owyang, C., (1989). Zinc absorption in human small intestine. Am. J. Physiol., 256:G87:G91. 8. Jackson, M.I., Jones, F.A., Edwards, R.H.T. et al. (1984). Zinc homeostasis in man: studies using a new stable isotope-dilution technique. Br. J. Nutr. 51:199-208. 9. Ziegler, E.E., Serfass, R.E., Nelson, S.E. et al. (1989). Effect of low zinc intake on absorption and excretion of zinc by infants studied with 70 Zn as extrinsic tag. J. Nutr. 119:1647-1653. 10. Lee, D.Y., Prasad, A.S., Hydrick-Adair, C., et al. (1993). Homeostasis of zinc in marginal human zinc deficiency: role of absorption and endogenous secretion of zinc. J. Lab. Clin. Med. 122:549-546. 11. Taylor, C.M., Bacon, J.R., Aggett, P.J. and Bremmer, I. (1992). Homeostatic regulation of zinc absorption and endogenous losses in zinc-deprived men. Am. J. Clin. Nutr. 53:755-763. 34

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35 12. Failla, M. L. and Cousins, R. J. (1978) Zinc uptake by isolated rat liver parenchymal cells. Biochim. Biophys. Acta 538: 435-444. 13. Pattison, S. E. and Cousins, R. J. (1986) Kinetics of zinc uptake and exchange by primary cultures of rat hepatocytes. Am. J. Physiol. 250: E677-E685. 14. Taylor, J. A. and Simons, T. J. (1994) The mechanism of zinc uptake by cultured rat liver cells. J. Physiol. 474: 55-64. 15. Mas, A. and Sarkar, B. (1991). Binding, uptake, and efflux of 65 Zn by isolated human trophoblast cells. Biochim. Biophys. Acta. 1092:35-38. 16. Ackland, M. L., Danks, D. M. and McArdle H. J. (1988) Studies on the mechanism of zinc uptake by human fibroblasts. J. Cell. Physiol. 135: 521-526. 17. Raffaniello, R. D., Lee, S. Y., Teichberg, S. and Wapnir, R. A. (1992) Distinct mechanisms of zinc uptake at the apical and basolateral membranes of Caco-2 cells. J. Cell. Physiol. 152: 356-361. 18. Bobilya, D. J., Briske-Anderson, M. and Reeves, P. G. (1992) Zinc transport into endothelial cells is a facilitated process. J. Cell. Physiol. 151: 1-7. 19. Menard, M. P. and Cousins, R.J. (1983) Zinc transport by brush border membrane vesicles from rat intestine. J. Nutr. 113:1434-1442. 20. Oestreicher, P. and Cousins, R. J. (1989). Zinc uptake by basolateral membrane vesicles from rat small intestine. J. Nutr. 119:639-646. 21. Tacnet, F., Watkins, D.W. and Rpoche, P. (1990). Studies of zinc transport into brush-border membrane vesicles isolated from pig small intestine. Biochem. Biophys. Acta 1024:323-330. 22. Tacnet, F., Lauthier, F., and Ripoche, P. (1993). Mechanisms of zinc transport into pig small intestine brush-border membrane vesicles. J. Physiol. (Lond.) 465:57-72 23. Cousins, R.J. and Leinhart, A.S. (1988). Tissue specific regulation of zinc metabolism and metallothionein genes by interleukin 1. FASEB J. 2:2884-90. 24. Henkin, R.I., Foster, D.M., Aamodt, R.L. and Berman, M. (1984). Zinc metabolism in adrenal cortical insufficiency: effects of carbohydrate-active steroids. Metabolism 33:491-501. 25. King, J.C., Hambidge, K.M., Westcott, J.L., Kern, D.L. and Marshall, D. (1994).Daily variatuions in plasma zinc concentrations in women fed meals at six hour intervals. J. Nutr. 124:508-16.

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36 26. Sharp, P., Tandy, S., Yamaji, S., Tennant, J., Williams, M. and Singh Srai, S.K. (2002). Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells. FEBS Lett. Jan 2;510(1-2):71-6. 27. Harris, D.S., Slot, J.W., Geuze, H.J. and James, D.E. (1992). Polarized distribution of glucose transporter isoform in Caco2 cells. Proc. Natl. Acad. Sci. 898:7556-60. 28. Fleet, J.C. and Wood, R.J. (1999). Specific 1,25(OH)2D3-mediated regulation of transcellular calcium transport in Caco-2 cells. Am J. Physiol. Apr;276(4 Pt 1):G958-64. 29. Fleet J.C.,Turnbull, J.C., Bourcier, M. and Wood, R.J. (1993). Vitamin D-sensitive and quinacrine-sensitive zinc transport in human intestinal cell line Caco-2. Am. J. Physiol. Gastrointest. Liver Physiol Jun 264: G1037 1045. 30. Cousins, R.J. (1996). Zinc. In: Present knowledge in nutrition. 7th ed. 29:293306. 31. Palmiter, R.D. and Findley, S.D. (1995). Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J. 14:639-49. 32. Wang K., Zhou, B., Kuo Y.M., Zemansky, J. and Gitschier J. (2002). A novel member of a zinc transporter family is defective in acrodermatitis enteropathica. Am. J. Hum Genet. 71(1):66-73. 33. Richards, M.P. and Cousins, R.J. (1975). Mammalian zinc homeostasis: requierement for RNA and metallothionein synthesis. Biochem. Biophys. Res. Commun. 64:1215-23. 34. Hoadley, J.E., Leinhart, A.S. and Cousins, R.J. (1988). Relationship of 65 zinc absorption kinetics to intestinal metalothionein in rats: effects of zinc depletion and fasting. J. Nutr. 118:497-502. 35. Oestreicher, P. and Cousins, R. J. (1989). Zinc uptake by basolateral membrane vesicles from rat small intestine. J. Nutr. 119:639-646. 36. Moore, J.B., Blanchard, R.K., McCormack, W.T. and Cousins, R.J. (2001). cDNA array analysis identifies thymic LCK as upregulated in moderate murine zinc deficiency before T-lymphocyte population changes. J. Nutr. 131(12):3189-96. 37. Markwell, M.A., Haas, S.M., Bieber, L.L. and Tolbert, N.E. (1978). A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87(1):206-10.

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37 38. Liuzzi, J.P., Bobo, J.A., Cui L., McMahon, R.J. and Cousins, RJ. (2003). Zinc transporters 1, 2 and 4 are differentially expressed and localized in rats during pregnancy and lactation. J Nutr. 133(2):342-51. 39. Dankert, J.R., Shiver, J.W. and Esser, AF. (1985). Ninth component of complement: self-aggregation and interaction with lipids. Biochemistry 24(11):2754-62. 40. Palmiter, R.D. (2004). Protection against zinc toxicity by metallothionein and zinc transporter 1. Proc. Natl. Acad. Sci 101(14):4918-4923. 41. Cragg, R.A., Christie, G.R., Phillips, S.R., Russi, R.M., Kury, S., Mathers, J.C., Taylor, P.M. and Ford, D. (2002). A novel zinc-regulated human zinc transporter, hZTL1, is localized to the enterocyte apical membrane. J. Biol Chem. 277(25):22789-97. 42. Kirschke, C.P. and Huang, L. (2003). ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J. Biol Chem. 2003 Feb 7;278(6):4096-102. 43. Wang, F., Kim, B.E., Dufner-Beattie, J., Petris, M.J., Andrews, G., and Eide, DJ. (2004). Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter. Hum. Mol. Genet. 13(5):563-71. 44. Schuerer-Maly, C.C., Eckmann, L., Kagnoff, M.F., Falco, M.T. and Maly, F.E. (1994). Colonic epithelial cells lines as a source of interleukin-8: stimulation by imflamatory cytokines and bacterial lipopolysaccharide. Immunology 81(1):85-91.

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BIOGRAPHICAL SKETCH Jeffrey A. Bobo was born in Hollywood, FL, in October of 1977. He attended South Fork High School in Stuart, FL, and began attending the University of Florida in the fall of 1996. He graduated with Bachelor of Science degrees in both nutritional sciences and zoology in the spring of 2000. He began work on his masters degree in nutritional sciences in the fall of 2002. 38


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Permanent Link: http://ufdc.ufl.edu/UFE0007019/00001

Material Information

Title: Characterization of Zinc Transporters in Caco-2 Cells
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0007019:00001

Permanent Link: http://ufdc.ufl.edu/UFE0007019/00001

Material Information

Title: Characterization of Zinc Transporters in Caco-2 Cells
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0007019:00001


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Full Text












CHARACTERIZATION OF ZINC TRANSPORTERS IN CACO-2 CELLS


By

JEFFREY ALAN BOBO
















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Jeffrey Alan Bobo















ACKNOWLEDGMENTS

First and foremost, I would like to thank my major professor, Dr. Robert J. Cousins

for the opportunity to work in one of the countries top research laboratories. I have

grown both as a scientist and a person and I am certain that the knowledge that I have

gained from this experience will assist me throughout my lifetime. I would also like to

thank my other committee members for their contributions and support towards my

project.

I would also like to thank my lab mates for their advice, expertise and

companionship: Ray Blanchard, Juan Liuzzi, Cal Green, Louis Lichten, Scott Rothbart,

Jennifer Moore, Jay Cao, Steve Davis, Ken Chiang, and Khanh Nguyen. I would also

like to extent a special thank you to Virginia Mauldin for her kindness, support and words

of motivation.

Last but not least, I would like to thank my wife, Dr. Rebecca Bobo for her love,

support and motivation, despite facing one of the toughest academic challenges herself.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iii

L IST O F TA B L E S .............. ................................................... ............... v.. .... ......v

LIST OF FIGURES ........................................ .............. vi

A B S T R A C T ..................................................................................................................... v iii

CHAPTER

1 IN T R O D U C T IO N ................................................. .............................................. .

2 LITER A TU RE REV IEW ................... ..............................................................2......

3 MATERIALS AND METHODS ........................................................7......

C ell C culture ................................................................................ ..........................7
Experimental Design ........................... ........... ...........................7
R N A Iso latio n ....................................................................................................... ... 8
Quantitative RT-PCR .......... ......... .. ........... ...............................8
P ro tein Iso latio n .................................................................................................... 9
Antibody Production ........................ .. ........... ...............................9
W western A n aly sis ........................................................................................................ 10
Electron M icroscopy ..... .. ................................................................................ 10
S statistic s ...................................................................................................... ....... .. 1 1

4 R E S U L T S ................................................................................................................. .. 1 3

R N A E expression ............... .. .................. .................... ............. ....... .. ............ 13
P ro tein ....................................................................................................... .......... 14
E lectro n M icro sco p y ................................................................................................... 15

5 D ISCU SSION ............................................................................ ....... ........ ............... 30

L IST O F R E FE R E N C E S ... ........................................................................ ................ 34

BIO GR APH ICAL SK ETCH .................................................................... ................ 38
















LIST OF TABLES


Table page

3-1 Peptides used to generate polyclonal antibodies to ZnT transporter proteins.......... 12

3-2 Peptides used to generate polyclonal antibodies to Zip transporter proteins ..........12
















LIST OF FIGURES

Figure page

4-1 ZnT1 zinc supplem entation tim e course ............................................. ................ 16

4-2 ZnT1 TPEN treatment time course ................................................... 16

4-3 ZnT4 zinc supplem entation tim e course ............................................. ................ 17

4-4 ZnT4 TPEN treatment time course ................................................... 17

4-5 ZnT5 zinc supplem entation tim e course ............................................. ................ 18

4-6 ZnT5 TPEN treatment time course ................................................... 18

4-7 ZnT6 zinc supplem entation tim e course ............................................. ................ 19

4-8 ZnT6 TPEN treatment time course ................................................... 19

4-9 ZnT7 zinc supplem entation tim e course ............................................. ................ 20

4-10 ZnT7 TPEN treatment time course ................................................... 20

4-11 Zipl zinc supplem entation tim e course..................................................... 21

4-12 Zipl TPEN treatm ent tim e course....................................................... ................ 21

4-13 Zip3 zinc supplem entation tim e course..................................................... 22

4-14 Zip3 TPEN treatm ent tim e course....................................................... ................ 22

4-15 Zip4 zinc supplem entation tim e course..................................................... 23

4-16 Zip4 TPEN treatm ent tim e course....................................................... ................ 23

4-17 Zipl mRNA expression in Caco-2 cells after 72 hr treatment with 50 ng/ml
IL l P ...................................................................................................... ........ .. 2 4

4-18 ZnT2 expression comparing vehicle control (100% EtOH) with treatment of 10
ng/m l calcitriol after 72 hrs ....................................... ....................... ................ 24

4 -19 Z ip l dot b lot .............. ................................................ .................... .. 2 5









4-20 Comparison Zipl protein expression after 24 h treatment of 5.0 gtM TPEN or
8 0 M zin c ............................................................................................................. 2 5

4-21 Effect of 50 ng/ml of IL j3 on Zip I protein after treatment for 24 h. ....................26

4-22 Effect of 10 ng/ml of calcitriol on ZnT2 protein expression...............................27

4-23 Electron micrograph of apical side of Caco-2 cell cells......................................28

4-24 Electron micrograph of control Caco-2 cells ......................................................28

4-25 Electron micrograph of ILl 3 treated Caco-2 cells.............................................29















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

CHARACTERIZATION OF ZINC TRANSPORTERS IN CACO-2 CELLS

By

Jeffrey Alan Bobo

August 2004

Chair: Robert J. Cousins
Major Department: Food Science and Human Nutrition

Zinc is an essential micronutrient that is tightly regulated in humans and other

animals and requires specialized mechanisms for its uptake, transport and excretion. Zinc

metabolism has been extensively studied in both man and rodents, and zinc transport has

received much attention with in vitro models. Numerous investigators have used isolated

cell types, including hepatocytes, fibroblasts, endothelial cells and intestinal cells to help

further characterize zinc transport. Of particular interest is the intestinal Caco-2 cell

model. Caco-2 cells, a human colonic tumor cell line, have been studied considerably in

the last decade as a model for drug, macronutrient and micronutrient absorption and

transport. There are several characteristics that render Caco-2 cells an ideal model for

absorption and transport. These cells can grow in monolayers and, upon confluency, the

cells differentiate and become polarized. They form tight junctions and possess a brush

border with functional brush border enzymes and thus resemble enterocytes.

Due to these inherent characteristics, we have focused on Caco-2 cells to

characterize the regulation of expression of the two zinc transporter gene families, ZnT









and Zip, under conditions of zinc deprivation and supplementation. The current

hypothesis is that ZnT family transporters decrease intracellular zinc concentration, while

Zip family transporters increase intracellular zinc concentration.

The human zinc transporter genes studied were ZnT1, 4, 5, 6, and 7, and Zip-1,

-3, and -4. The Caco-2 cells were treated with TPEN, a cell permeable zinc chelator, at

1.25, 2.5 or 5.0 riM, zinc sulfate at 40 or 80 riM, ILl 0, or calcitriol. Transporter

expression was measured by quantitative real-time PCR, western blotting and

immunohistochemistry. The results from this study demonstrate that some transporters

from both families show differential regulation of expression based on treatment, while

others were refractory to change due to zinc status. ZnT1, 4, 7 and Zip4 expression was

decreased by zinc deprivation, while ZnT-6 was up-regulated. Zinc restriction

up-regulated Zipl, while Zip3 was down-regulated. Zipl was upregulated by IL1 0 while

ZnT2 was downregulated by calcitriol. Overall, the results suggest genes from both zinc

transporter families are differentially expressed in Caco-2 cells in a mode consistent with

maintenance of cellular zinc homeostasis in zinc restriction and supplementation, and

certain zinc transporter genes are affected by physiological stimuli.














CHAPTER 1
INTRODUCTION

Zinc is intimately involved in many metabolic processes in the body including

DNA replication, immune function and protein-protein interaction. Subsequently is it an

essential micronutrient that is regulated under tight control in humans and other animals

and requires specialized mechanisms for transport (1, 2). There are a number of zinc

transporters that are involved in the transport and metabolism of zinc either through

efflux or influx (3, 4) and these transporters are from two distinct gene families, ZnTs

and ZiPs. Over the last several years numerous studies have been conducted and there

have been several discoveries of additional transporters in both of these families. The

purpose of this study is to characterize the various zinc transporters from the ZnT and ZiP

families found in Caco-2 cells and to further understand the mechanisms by which zinc is

absorbed and transported in Caco-2 cells.














CHAPTER 2
LITERATURE REVIEW

Zinc is an essential divalent cation that is required for many biochemical processes

in mammals. It is a strong Lewis acid and does not demonstrate any redox chemistry,

which is why it is involved in many biological processes (1). The biochemical roles of

zinc can be divided into three different categories, catalytic, structural and regulatory.

Catalytically zinc has a concentrated 2+ charge, which results in high electron affinity and

makes it a good attacking group. There are over 300 known zinc metalloenzymes, which

require zinc for function, some of which are involved in gene transcription, i.e. RNA

polymerases. The structural role of zinc is also a result of its chemistry and is

demonstrated through zinc finger proteins. Zinc fingers are regions in proteins that have

a structure of cysteine and histidines to achieve a unique structure that allows the binding

of zinc. These proteins have a variety of functions that include transcription factors, RNA

binding proteins and protein-protein interactions. The regulatory role of zinc is seen in

the regulation of metal response elements, or MREs. MREs are located in the promoter

regions of regulated genes and can interact with metal-binding transcription factors,

MTFs. MTFs are zinc finger proteins that acquire zinc from the cytosol, translocate to

the nucleus and interact with MREs to stimulate transcription (1).

Due to the wide variety of essential roles mentioned above, it is easy to see why

homeostatic control of zinc levels is important. Even a minor zinc deficiency could

potentially cause a decrease in one of the functions of this micronutrient that could

influence health. Clinical manifestations due to mild to severe zinc deficiency range









from alopecia, acrodermatitis, diarrhea, impaired wound healing and immunity, as well as

growth retardation, and hypogonadism. Therefore it is important to understand the

absorption of this essential micronutrient from the gastrointestinal tract.

Zinc is absorbed throughout the small intestine, but studies suggest that the

duodenum and the jejunum are the primary sites (5, 6, 7). In order to maintain zinc

homeostasis, a balance must exist between absorption of dietary zinc and endogenous

secretions through adaptive regulation to dietary zinc (8). The intestine is the key organ

in maintaining zinc balance through absorption and endogenous excretion. It has been

demonstrated in humans and animals that a restriction of dietary zinc results in increased

uptake and efficiency of intestinal absorption (9-11).

Zinc transport has received much attention through research with in vitro models.

Numerous investigators have used isolated cell types, including hepatocytes (12-14),

placental trophoblasts (15), fibroblasts (16), intestinal cells (17), endothelial cells (18),

and membrane vesicles (19-22) to help further characterize zinc transport. Studies using

radioisotopes have identified and given insight into zinc processing and pathways that

result in the overall homeostatic control of zinc levels in the body (3). Though the models

differ, there are several distinct characteristics that can be concluded. Zinc transport is a

time-, concentration-, pH-, and temperature-dependent process, with some evidence

demonstrating saturable and nonsaturable components (2). Metabolic studies have shown

that dietary zinc intake influences intestinal absorption, while certain hormones,

cytokines and growth factors influence zinc metabolism in specific certain organ systems

(23-25).









Since the intestine is the key regulator of zinc homeostasis, intestinal cell models

are of particular interest. Caco-2 cells, a human colonic tumor cell line, have been

studied considerably in the last decade as a model for drug, macronutrient, and

micronutrient absorption and transport. There are several characteristics that render

Caco-2 cells an ideal model for absorption and transport. These cells grow in monolayers

and upon confluency, the cells differentiate and become polarized. They form tight

junctions and possess a brush border with functional brush border enzymes and thus,

resemble enterocytes (17).

Transport and absorption has been shown to be regulated in Caco-2 cells for several

nutrients including iron, glucose, calcium and zinc. Treatment of Caco-2 cells with high

concentrations of iron caused DMT1, an iron transporter protein typically located at the

apical membrane to decrease while total cellular DMT1 levels remained steady (26).

These results demonstrate that in order to decrease iron absorption the transporter protein

is able to translocate based on the needs of the cell. It has also been shown that different

isoforms of the glucose transporters (GLUT) are differentially localized (27). Fleet and

Wood (28) used Caco-2 cells to investigate the effects of active vitamin D on calcium

transport. They showed that calcium transport increases upon addition of calcitriol to the

medium.

Focusing on zinc transport, Raffaniello et al. (17) demonstrated there were two

distinct pathways at the apical and basolateral sides in Caco-2 cells as well as a

vesicularly mediated component. Additionally, Fleet et al. (29) noticed that Caco-2 cells

treated with calcitriol increased zinc transport by 159% and that this increase was not due

to lysosome-mediated zinc transport. The molecular mechanisms of zinc transport and









absorption are not fully known but involve brush border transport, intracellular transport

and basolateral transport (30).

In the last decade, there have been numerous studies that have provided evidence

that there are a number of transporter proteins that are involved in zinc transport in

mammals. The first zinc transporter gene cloned was ZnT1 by Palmiter and Findley (31).

Since its discovery, several other zinc transporters have been discovered and

characterized. In fact, there are actually two distinct gene families of zinc transporters,

ZnTs and Zips (3). The current hypothesis is that ZnT proteins reduce intracellular zinc

either by efflux from the cell or by sequestering zinc within vesicles. The ZiP proteins

are thought to increase intracellular zinc through extracellular uptake or by transport from

vesicles into cytoplasm (3).

Brush border uptake characteristics by enterocytes suggest both saturable and

nonsaturable components at low and high luminal zinc concentrations, respectively (30).

The transporters thought to be located apically, and thus responsible for luminal uptake or

cellular efflux are Zipl, 2, 4 and ZnT5 (3). Interestingly, it was found by Wang et al.

(32) that a mutation in the gene that encodes for Zip4 is the cause for the disease

acrodermatitis enteropathica, in which patients suffer from a severe zinc deficiency due

to malabsorption of dietary zinc.

It is known that protein synthesis is required for the downregulation of zinc

absorption under high concentrations of zinc (33) and that metallothionien (MT) is a

major zinc binding protein in intestinal cells. MT has been used as the sentinel gene for

cellular zinc status. Its expression is increased by high dietary zinc and decreased with

lower zinc intake. The current hypothesis is that MT acts as a buffer for intracellular zinc









(34) so it controls how much zinc can be transferred to the basolateral and apical surfaces

for either transport into circulation or back into the lumen, respectively. Zinc absorption

decreases as MT synthesis rises in response to dietary zinc. As mentioned above, there is

also evidence of a vesicularly or lysosomal mediated component of zinc transport in

Caco-2 cells (17, 29). At this time the function of this phenomenon is not known,

however, zinc transport in to and out of the vesicles could be a component of the

absorption process. The transporters that are thought to be located vesicularly are ZnT2,

4, 6, and 7 and Zip I and 3.

Evidence suggests that basolateral zinc transport is comprised of both saturable and

nonsaturable components in both directions (34, 35). Basolateral transfer appears to be

linear and zinc concentration dependent. Endogenous zinc returns to the intestine as

enterocytes slough off into the lumen or as pancreatic secretions. The zinc transporters

that are thought to be involved in zinc trafficking by enterocytes have not been identified.

The purpose of the current study is to gain insight into the regulation of zinc

homeostasis by the intestine using Caco-2 cells as a model in order to see the changes in

expression of the zinc transporter genes under conditions of zinc deprivation, zinc

supplementation and other physiological factors, including IL1 P3 and calcitriol.














CHAPTER 3
MATERIALS AND METHODS

Cell Culture

Caco-2 cells were obtained from American Type Culture Collection (Rockville,

MD). They were maintained in DMEM under standard conditions: 37 C and 5% CO2.

Cells were in medium containing 10% v/v FBS. Once experimental design was planned,

cells were seeded into the desired number of T-25 culture flasks at approximately 3.5 x

105 cells/cm2. The following day the meduim was changed to DMEM with 5% v/v FBS

and MITO+ cell differentiation/ serum extender supplement (BD Biosciences) in order to

induce differentiation of the Caco-2 cells. The cells were then allowed to differentiate for

five to seven days with medium changes every 48 h prior to experimentation.

Experimental Design

Cells were treated in order to mimic conditions of either a zinc deprived state or

zinc excess. Zinc deprivation was produced using (TPEN), a cell permeable zinc chelator

with a high affinity for zinc (Kd = 10-16M). The medium with 5% v/v FBS has a zinc

concentration of approximately 1.54 riM. Since TPEN has a 1:1 binding capacity with

zinc, three different TPEN concentrations were used: 1.25, 2.5 and 5.0 riM, in order to

mimic a moderate to severe zinc deficiency. Zinc sulfate was used to supplement the

medium with excess zinc (40 and 80 [aM). For both treatment conditions, TPEN and

excess zinc, a time course was performed in order to discover the largest change in

transporter transcript expression. The time points used were 0, 4, 8, 12, 24, and 48 h.









Cells were also treated with IL1B, a proinflamatory cytokine, at 50 ng/ml or calcitriol,

the hormonal form of vitamin D, at 10 ng/ml for 24 h.

RNA Isolation

For each T-25 culture flask, one ml of TriPure Isolation Reagent (Roche Molecular

Biochemicals, Indianapolis, IN) was added after removal of the medium. The cells were

then scraped from the flask and vigorously triturated for 1 min. The sample was then put

into microfuge tubes and stored at -80 C until all RNA was harvested for the entire time

course. Samples were then thawed, 0.2 ml of chloroform was added, followed by

vigorous shaking for 15 sec. The samples were then centrifuged for 10 min at 4 C at

12,000 x g and the clear aqueous layer was transferred to a new sterile tube and 0.5 ml of

isopropyl alcohol was added to precipitate RNA. Samples were gently mixed and left

sitting at room temperature for 10 min and then centrifuged again for 10 min at 12,000 x

g. RNA pellets were then washed 3 times with 75% v/v ethanol followed by

centrifugation at 7500 x g for 5 min. Following ethanol washes, RNA pellets were

resuspended in 200 all of diethyl carbonate (DEPC)-treated water. Purity and

concentration were determined spectrophotometrically as described by Moore et al. (36).

Each sample was then aliquoted (50 [il) and treated with DNase I (Ambion, Austin, TX)

to eliminate DNA contamination.

Quantitative RT-PCR

Primers and probes for each transporter gene were designed using Primer Express

software (Applied Biosystems, Foster City, CA) based on sequence information for each

gene. The relative quantity of RNA was determined by using 18S rRNA (primer and

probes designed by Applied Biosystems) for normalization. A standard curve was

generated using 1:10 dilutions of total RNA starting at lOng/ml and producing a 4- to 5-









log range for each determination. The ABI Model 5700 Sequence Detection System was

used to fluorometrically measure amplification of PCR products for each transporter gene

transcript.

Protein Isolation

Cells were grown and treated as described above except T-75 culture flasks were

used. The standard treatment time was 24 h. Cells were rinsed twice with PBS prior to

the addition of 5 ml of a modified RIPA buffer containing: l%Triton x-100, 0.5% w/v

sodium deoxycholate, 1% v/v SDS in PBS with protease inhibitor cocktail P-2714

(Sigma, St. Louis, MO). Cells were scraped from the flasks and then triturated with a 25

gauge needle in order to shear the nucleic acids in the sample. The protein concentration

of the total cell preparation was then determined by colorimetric assay (37). A standard

curve was established using bovine serum albumin and the absorbance was read at 650

nm. Linear regression was then used to determine protein concentration.

Antibody Production

Peptides (Biosynthesis Incorporated, Lewisville, TX), shown in Table 1 and 2,

were coupled to KLH (Pierce, Rockford, IL) and used to generate polyclonal antibodies,

as described previously (38). Total IgG was isolated by the precipitation method (39).

Caprylic acid was added to the serum (pH 5.0 with acetic acid) in order to precipitate

serum proteins other than IgGs. Serum was centrifuged for 30 min at 35,000 x g.

Supernatant was removed and ammonium sulfate was added (1:1, v/v) to precipitate the

total IgG fraction. Some of the total IgG fractions were then affinity purified (AP)

(Sulfolink; Pierce). Specificity of the affinity purified antibodies and IgG fractions were

determined by comparing signals from pre-immune serum, total IgG and where

applicable, affinity purified IgG and flowthrough effluent from the affinity









chromatography. The methods are comparable to those reported by McMahon and

Cousins (4).

Western Analysis

Equal amounts (30 pg) of total cellular protein were resolved by 10% SDS-PAGE

gels and transferred to PVDF (Immobilon; Millipore, Bedford, MA) in standard Towbin

transfer buffer (20% w/v methanol). Blots were stained with Ponceau Red to verify

equal protein loading and then destined. Membranes were wetted with methanol and

then placed in Milli-Q water to allow complete wetting, followed by blocking in 5% w/v

nonfat dry milk (Carnation) in PBS-T (0.025% v/v Tween 20). Membranes were washed

for 5 min in PBS-T and then incubated for 1 h in PBS-T with 1:100 of whole serum, 10-

20 [tg/ml of total IgG or 2-4 pg/ml of affinity purified IgG; followed by three 5 min

washes with PBS-T. Membranes were incubated with anti-rabbit IgG secondary antibody

conjugated to alkaline phosphatase (Amersham Biosciences, Piscataway, NJ) for Ih,

washed 3 x for 5 min each and then viewed with a chemiluminescent reagent for

detection (Amersham Biosciences) using fluorescence imaging (Molecular Dynamics

Storm 840 Imager).

Electron Microscopy

Caco-2 cell monolayers were grown and allowed to differentiate as previously

mentioned on fibrillar collagen I inserts (Becton Dickinson Labware, Bedford, MA).

Cells were rinsed 2 x with sterile PBS and fixed in a 0.1 M sodium cacodylate buffer

containing 4 % w/v paraformaldehyde and 0.5 % w/v glutaraldehyde for 24 hrs at 4C at

which time the fixative was changed to a 0.1 M cacodylate buffer. Cells were stored in

this buffer at 4C until further processing.









Inserts with attached cells were then dehydrated through a series of ethanol washes

for 15 min each. The first wash was 15% v/v ethanol followed by 20%, 25%, 30%, 50%,

70%, and 90%. The next incubation was in 90% ethanol mixed 1:1 with the LR White

(Electron Microscope Sciences, Hatfield, PA) embedding medium for 10 min, followed

by a 10 min incubation in 100% LR White. The membranes were then excised from the

insert chamber and then cut in half and once again incubated in 100% LR White for 20

min, then one h and then left rotating overnight at room temperature. The next morning

each 12 membrane was placed into fresh LR White and then kept in a 60C oven

overnight in gelatin capsules to harden. Extra embedding material was trimmed away in

order to cut sections perpendicular to the plane of the membrane. Sections were cut 80-

90 nm thick and mounted on formvar nickel coated 200 mesh grids (Electron Microscope

Sciences, Hatfield, PA).

Grids were blocked in 1% w/v BSA in sterile filtered PBS for 15 min and rinsed in

PBS-T (0.025% v/v Tween 20) for 1 min. Grids were incubated in PBS-T containing

primary antibody diluted about 1:100 and placed in a dark, damp chamber for 1 h. Grids

were washed 3 x in PBS-T for 3 min each followed by 2 washes in PBS for 3 min.

Samples were then placed in secondary, immunogold labeled (18 nm) antibody solution

(1:50) (Jackson Immunoresearch, West Grove, PA) and incubated for 1 h, followed by 3,

5 min washes in PBS, a 5 min incubation with Trumps and 4, 5 min washed in Milli Q

Water and one 15 s jet wash. Specimens were viewed with a Hitachi H-7000

Transmission Electron Microscope (ICBR EM Core Laboratory, University of Florida).

Statistics

Each data point represented throughout the project was a replicate of at least three

different trials (n=3), unless otherwise noted. A one-way ANOVA was conducted in









order to determine if there were any differences between data groups. The post hoc test

was performed using the Student Newman-Keuls multiple comparison test. Differences

were considered significant with a p-value of equal to or less than 0.05.

Table 3-1 .Peptides used to generate polyclonal antibodies to ZnT transporter proteins.
Transporter Peptide
m/rZnTI GTRPQVHSGKE

m/rZnT2 CHAQKDSGSHP

m/r/h/ZnT4 MQLIPGSSSKWEE

m/hZnT5 SPSKRGQKGTLI

mZnT6 VAPNVLNFSDHHVIP

m/h/rZnT8 QKPVNKDQCP


Table 3-2. Peptides used to generate polyclonal antibodies to Zip transporter proteins.
Transporter Peptide
m/hZIP1 RSGANHEASASG


m/rZIP2 EEEWGGTHAFGFH


m/rZIP3 CAGLRLRELGRPG


AEETPELLNPETRRL


mZIP4














CHAPTER 4
RESULTS

RNA Expression

Zinc transporter transcripts that were detected in Caco-2 cells using quantitative

PCR (Q-PCR) were ZnT1, 4, 5, 6, and 7 and Zip 1, 3, and 4. Transcript data at each time

point are presented on a relative basis where vehicle control expression levels are equal to

one. ZnT1 expression increased 2-fold by 4 h in response to supplemental zinc (Zn+)

(Fig 4-1). Levels remained elevated for 24 h and then returned to basal conditions. ZnT1

mRNA levels were greater at 80 [M Zn than 40 [M Zn. Figure 4-2 shows that ZnT1

expression decreased rapidly with all concentrations of TPEN. The response to zinc

deprivation (Zn-) with TPEN chelation was greatest after 4 h. Figures 4-3 illustrates that

ZnT4 decreased steadily starting at 12 h with 80 [iM zinc treatment reaching 2-fold at 48

h, while ZnT4 mRNA levels decreased only in the more severe Zn- conditions, reaching a

decrease of nearly an 8-fold after 24 h of treatment with 5.0 [M TPEN (Fig 4-4). ZnT-5

transcript levels were refractory to any significant changes for either Zn+ and Zn-

through out the time courses except a 9-fold decrease at 48 h with 5.0 [M TPEN (Figs 4-

5 & 4-6). However, the 48 h time point with 5.0 [M TPEN treatment was not considered

valid because cells were more than 50% apoptotic by trypan blue exclusion (data not

shown). ZnT6 transcript expression was refractory to change under zinc supplementation

(Fig 4-7), but treatment 5.0 [M TPEN treatment increased ZnT6 expression up to 2-fold

by 12 h (Fig 4-8). Conditions of zinc excess had no effect on ZnT7 expression levels

(Fig 4-9). In contrast, conditions of mild zinc deprivation (1.25 [M TPEN) had no effect









on ZnT7 expression, while 2.5 [M TPEN increased expression 2-fold at 12 h and 48 h

with 2.5 pM TPEN and 5.0 iM TPEN decreased transcript levels 3-fold after 24 h (Fig 4-

10).

Zinc supplementation caused a 2-fold decrease in expression of the Zip transcript

at 12 h (Fig 4-11). TPEN treatment of 2.5 and 5.0 itM caused over a 2-fold increase in

Zipl expression starting at 12 h and continuing through 24 h (Fig 4-12). Zip3 transcript

expression was not significantly altered by zinc excess, however zinc deprivation caused

a steady decrease in expression starting at 8 h and resulting in an 8-fold decrease by 24 h

(Figs 4-13 & 4-14). Zip4 also had no significant changes in transcript expression with

zinc supplementation, while zinc deprivation decreased Zip4 expression almost 2-fold

(Fig 4-15 & 4-16).

Two of the zinc transporters, one from each gene family, Zip 1 and ZnT2, were

responsive to the two physiological stimuli tested. Zipl was the only transporter that was

regulated by IL13, increasing RNA expression almost 5-fold over the controls as seen in

Figure 4-17. ZnT2 was the only transporter that changed mRNA levels when cells were

first treated with 10 nmol calcitriol for 72 h compared to control cells. Expression

decreased approximately 3-fold when compared to control cells (Fig 4-18).

Protein

Preimmune serum (PIS), affinity purified IgG (AP), and the flow through (FT,

affinity chromatography effluent) were used to determine the specificity of the antibodies

utilized for further examination of Zipl and ZnT2 transporters (Fig 4-19). Protein

expression for Zipl comparing Zn+ to Zn- shows that zinc deprivation increased Zipl

expression while zinc excess decreased protein expression by the 24 h time point (Fig 4-






15


20). In addition, treatment with IL1 0 caused a 3.5-fold increase in Zipl protein

expression. ZnT2 protein expression was decreased nearly 2-fold with 10 nmol/ml of

calcitriol by the 24 h time point (Figure 4-21).

Electron Microscopy

Electron micrographs (Fig 4-23 & 4-24) illustrate that Zipl is located vesicularly

throughout cytoplasm and to a smaller extent to the brush border membrane. Figure 4-25

demonstrates that upon treatment with IL1 0, Zipl in upregulated, which is in agreement

with mRNA and western data.










ZnT-1 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course


ZnSO,

* 40 uri
o- 00 LriJ


Time (hrs)


Figure 4-1. ZnT1 zinc supplementation time course. Values at each time point are
relative to vehicle control (vehicle control=l1 at each time point). Data
represented by mean + standard deviation (n=3).


ZnT-1 TPEN Time Course


10



1



0.1


0.01


TPEN (uM)

O [1.25]
O [2.5]
[5.0]


Hours


Figure 4-2. ZnT1 TPEN treatment time course. Values at each time point are relative to
vehicle control (vehicle control=l1 at each time point). Data represented by
mean standard deviation (n=3).


rtt 1. CP 0








ZnT-4 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course


Time (hrs)


Figure 4-3. ZnT4 zinc supplementation time course. Values at each time point are
relative to vehicle control (vehicle control=l1 at each time point). Data
represented by mean + standard deviation (n=3).

ZnT-4 TPEN Time Course


1


0.1


0.01


TPEN (uM)
O [1.25]
O [2.5]
[5.0]


Hours


Figure 4-4. ZnT4 TPEN treatment time course. Values at each time point are relative to
vehicle control (vehicle control=l1 at each time point). Data represented by
mean standard deviation (n=3).


ZnSO,
* 40 ur,
0o 00 1rA


Sz r ^ i


S111






18


ZnT-5 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course


ZnSO,
* -40 uM


Time (hrs)

Figure 4-5. ZnT5 zinc supplementation time course. Values at each time point are
relative to vehicle control (vehicle control=l1 at each time point). Data
represented by mean standard deviation (n=3).

ZnT-5 TPEN Time Course


10





I




0.1


TrPEN (uM)

o [1.25]
o [2.5]
[5.0]


Hours


Figure 4-6. ZnT5 TPEN treatment time course. Values at each time point are relative to
vehicle control (vehicle control=l1 at each time point). Data represented by
mean standard deviation (n=3).


-~-~


iI T









ZnT-6 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course


ZnSO,

* 40 ur,
[o 00 1rA


Time (hrs)


Figure 4-7. ZnT6 zinc supplementation time course. Values at each time point are
relative to vehicle control (vehicle control=l1 at each time point). Data
represented by mean standard deviation (n=3).


ZnT-6 TPEN Time Course


TPEN (uM)

[1.25]
0 [2.5]
[5.0]


Hours


Figure 4-8. ZnT6 TPEN treatment time course. Values at each time point are relative to
vehicle control (vehicle control=l1 at each time point). Data represented by
mean standard deviation (n=3).


I~ -~ ~


V I










ZnT-7 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course


10

Z ZnSO,

u 40 -uM
a 1 2 T I, P. _-


0.
x


E 0.1
4 8 12 24 48
Time (hrs)


Figure 4-9. ZnT7 zinc supplementation time course. Values at each time point are
relative to vehicle control (vehicle control at each time point). Data
represented by mean + standard deviation (n=3).


ZnT-7 TPEN Time Course


10
U)


-,
e -

Cu


TPEN (uM)

[1.25]
[2.5]
*[5.0]


Hours

Figure 4-10. ZnT7 TPEN treatment time course. Values at each time point are relative to
vehicle control (vehicle control=l1 at each time point). Data represented by
mean standard deviation (n=3).


- _i










ZiP-1 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course


ZnSO,

* 40 ur,
0o 00 1rA


Time (hrs)


Figure 4-11. Zipl zinc supplementation time course. Values at each time point are
relative to vehicle control (vehicle control=l1 at each time point). Data
represented by mean standard deviation (n=3).


ZiP-1 TPEN Time Course


TPEN (uM)

O [1.25]
D [2.5]
[5.0]


Hours


Figure 4-12. Zipl TPEN treatment time course. Values at each time point are relative to
vehicle control (vehicle control=l1 at each time point). Data represented by
mean standard deviation (n=3).









ZiP-3 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course


ZnSO,
* 40 u4,,
0O 0 LIrP,


Time (hrs)


Figure 4-13. Zip3 zinc supplementation time course. Values at each time point are
relative to vehicle control (vehicle control=l1 at each time point). Data
represented by mean standard deviation (n=3).

ZiP-3 TPEN Time Course


TPE N (uM)

0 [1.25]
D [2.5]
[5.0]


Hours


Figure 4-14. Zip3 TPEN treatment time course. Values at each time point are relative to
vehicle control (vehicle control=l1 at each time point). Data represented by
mean standard deviation (n=3).


I i-i "~ +~ -~


11










ZiP-4 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course


ZnSO,

* 40 ur,
o0 LC' 1


Time (hrs)


Figure 4-15. Zip4 zinc supplementation time course. Values at each time point are
relative to vehicle control (vehicle control=l1 at each time point). Data
represented by mean standard deviation (n=3).


ZiP-4 TPEN Time Course


TPEN (uM


O [1.25]
E [2.5]
* [5.0]


Hours

Figure 4-16. Zip4 TPEN treatment time course. Values at each time point are relative to
vehicle control (vehicle control=l1 at each time point). Data represented by
mean standard deviation (n=3).


MINE= L





































Figure 4-17. Zipl mRNA expression in Caco-2 cells after 72 hr treatment with 50 ng/ml
ILl p.


ZnT2 mRNA Expression after 72 hr Treatment with
Calcitriol


< 1.2
z

II-
a 0.8

0.6

0.4
0.2 -


*) 0
Control Calcitriol


Figure 4-18. ZnT2 expression comparing vehicle control (100% EtOH) with treatment of
10 ng/ml calcitriol after 72 hrs. Note: These data (calcitriol mRNA
experiment only) reflect a pilot experiment that was conducted (n=l). Further
testing would be appropriate to confirm these results)


Caco 2 Cells hZiP-1 mRNA IL-B Treatment

7

6



d.4
SControl
3 IL-1 B

"o 2









I
hi


*


A. B. C. D.
Figure 4-19. Zipl dot blot: A. Immune serum and B. Pre-immune serum, C & D.
Western blot: C. Affinity purified antibody and D. Flow through.



ZiP-1 Protein in Caco-2 Cells:


3


2.5


2


1.5


1


0.5


5uM TPEN


80uM Zn


Treatment

Figure 4-20. Comparison Zipl protein expression after 24 h treatment of 5.0 [M TPEN
or 80 L M zinc (n=3).











Zip1 Protein ILl B Exp.


5

4.5

4

3.5

,- 3
E
0 2.5
2

1.5
1

0.5

0
Control IL1 B

Figure 4-21. Effect of 50 ng/ml of ILl13 on Zipl protein after treatment for 24 h (n=3).












ZnT2 Protein: Calcitriol treatment


1.4 -








E 0.8 -
0






0.2


0 !


Control


Calcitriol


Figure 4-22. Effect of 10 ng/ml of calcitriol on ZnT2 protein expression (n=3).









F- :, ^ ^

.4'
1kI-! -

i^^^|


Figure 4-23. Electron micrograph of apical side of Caco-2 cell cells. A. and B. are
negative controls. (No primary antibody was used, but all other
immunolabeling steps were conducted). C. and D. Represent control cells that
were immunogold-labeled with polyclonal Zip antibody along the apical
membrane. The arrows are pointing to 18 nm gold particles attached to
secondary antibody.


Figure 4-24. Electron micrograph of control Caco-2 cells. A. Shows positive labeling
between nucleus (left side) and apical membrane. B. Shows some positive
labeling just outside the nucleus, possibly representing the rough endoplasmic
reticulum.

































Figure 4-25. Electron micrograph of ILl 3 treated Caco-2 cells. A. and B. Show
considerable labeling throughout the cytoplasm and along the brushborder. C.
Represents labeling around a junction between two cells. D. demonstrates
labeling around the nucleus' of two cells.














CHAPTER 5
DISCUSSION

The results from this project demonstrate that there is differential regulation of

expression of the zinc transporters from both gene families, Zip and ZnT, in Caco-2 cells.

These data give us further insight into the mechanistic control of zinc absorption,

however one must consider that this is an in vitro model using a transformed cell line.

ZnT1 expression was consistent with current literature as far as its response to low

and high zinc levels in both cell culture and animal studies (4). ZnT1 had a quick

response (within 4 hrs) to both zinc deprivation and supplementation. Interestingly, the

lowest TPEN treatment (1.25 gM), used to mimic a mild deficiency, decreased ZnT1

expression by 2-fold. These data indicate that ZnT1 regulated in a way that allows it to

rapidly adapt to environmental conditions and may be part of the first defense against

environmental changes in the zinc supply. Furthermore, these data support that ZnT1

functions as a cellular zinc exporter which downregulates to conserve zinc. In addition,

Palmiter (40) has shown there to be an interaction between ZnT1 and metallothionein, the

sentinel gene for cellular zinc status, which further suggests ZnT1 has a role in adaptation

to differential zinc concentrations within cells.

ZnT4 was essentially refractory to zinc supplementation. Similarly, only severe

zinc depletion resulted in ZnT4 downregulation. Assuming that ZnT4 is vesicular and

exports zinc from the cytoplasm into vesicles, that the two lower TPEN treatments

produce ZnT4 is downregulation indicates that this transporter may conserve zinc that is









left in the cell. Alternatively, the function of ZnT4 may not be critical for survival and

begins to shut down during a severe zinc deficiency.

There was no change in ZnT5 within the 48 h time period examined which could

indicate that ZnT5, shown to be located apically (41) in intestinal cells is refractory to

change based on nutrient status. However, Cragg et al. (41) showed that ZnT5 increased

after treatment of Caco-2 cells with 100 gM zinc for 7 d. Perhaps ZnT5 expression

changes only during chronic conditions of zinc excess.

ZnT6 significantly increased only at 12 h with 2.5 and 5.0 gM TPEN. This could

signify that ZnT6 may serve to sequester zinc once the cell detects that levels are

dropping in order to conserve zinc for vital function. This hypothesis is in agreement

with the observation that there is no change in ZnT6 with zinc supplementation.

ZnT7 distribution is in small intestine, liver and spleen and to a lesser extent in the

kidney lung and brain. It is located vesicularly and functions to accumulate zinc into the

golgi apparatus (42). In this study, zinc supplementation had no effect on ZnT7

expression. Since ZnT7 mRNA upregulates with mild TPEN treatment, our results agree

with the conservation (accumulation) concept. However, as shown with ZnT5 and ZnT6,

5.0 gM TPEN decreases ZnT7 expression and may demonstrate a level of functional

priority necessary for cell survival.

The increase in Zipl, which was found localized apically as well as vesicularly,

follows the current hypothesis that this transporter's function is to increase the

intracellular zinc concentration (3). From the data collected, Zip 1 is thought to be the

major importer of cellular zinc, or at least the importer that is the most regulated in Caco-

2 cells. This information somewhat agrees with data that Wang et al. (43) found in









transfected HEK 293 cells showing that under Zn- conditions, Zipl localizes to the

plasma membrane.

The location of Zip3 in intestinal cells is not known, but Wang et al. (43) showed

that Zip3 cDNA stimulates zinc uptake when transfected into HEK293 cells and that the

subcellular distribution of Zip3 is zinc regulated. They found that Zip3 expression at the

plasma membrane increased only after more severe zinc deprivation compared to Zip 1.

Zip3 mRNA expression was significantly downregulated by severe zinc deprivation in

this study, suggesting that its function in the intestine may differ from that in transfected

kidney cells. The function of Zip3 in Caco-2 cells will be more clear once its

intracellular localization is established.

Zip4 is the gene that is thought to be the most important for zinc absorption since it

is responsible for the disease Acrodematitis Enteropathica. In this genetic disease

patients exhibit zinc malabsorption and suffer from a severe zinc deficiency due to a

mutation of the Zip4 gene. The results presented in this project differ with respect to

Zip4 expression in mice, where zinc deficiency produces a marked increase in expression

(Liuzzi unpublished data).

There are several reasons transporter expression may vary among cell types and

between cell lines. Caco-2 cells, though a good model, have such limitations. The first

and most obvious reason is that these cells are not enterocytes. They are transformed

colonic cells that have enterocyte-like properties. Additionally, this study has shown that

Zip and ZnT2 are responsive to IL1 P3 and vitamin D treatments, respectively. Ill P was

used because it was shown to induce L8 secretion in Caco-2 cells (44) and we were

interested to see if there was any change in zinc transporter expression due to an induced









immune response as might be seen in inflammatory bowel disease. Calcitriol was shown

to increase zinc transport in Caco-2 cells by 150% by Fleet et al. (29) so we were

interested to see which transporters might be responsible for the increased zinc transport.

The reduction in ZnT2 expression by calcitriol suggests this hormonal form of vitamin D

might stimulate zinc absorption by decreasing intestinal retention. ZnT2 has been related

to intestinal zinc storage (38). The fact that physiological and not just nutrient stimuli

effect regulation shows that there may be several different signaling systems responsible

for regulation of zinc transporter expression in an intact animal.

None of the Zip transporters that were found in Caco-2 cells, Zip 1, 3, and 4 were

found to be highly regulated by zinc supplementation. The data from this project reflect

the first model using Caco-2 cells to survey an array of zinc transporters and may serve as

a springboard for future research. This current project uses RNA and some protein data

to further understand the functions of these transporters based on observed expression

changes due to certain external stimuli, nutrient or physiological. It shows that there are

several levels of differential expression for each of the transporters. Continued study

using this cell model to predict function and roles should lead to a greater understanding

of transporter function. The fact that many of these transporters respond directly to a

nutrient excess or deprivation lean to the notion that they exhibit zinc responsiveness

which is an elementary mechanism of nutrient regulation.
















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BIOGRAPHICAL SKETCH

Jeffrey A. Bobo was born in Hollywood, FL, in October of 1977. He attended

South Fork High School in Stuart, FL, and began attending the University of Florida in

the fall of 1996. He graduated with Bachelor of Science degrees in both nutritional

sciences and zoology in the spring of 2000. He began work on his master's degree in

nutritional sciences in the fall of 2002.