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.
iii
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
LIST OF TABLES
page
Table
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
LIST OF FIGURES
page
Figure
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 β. .........................................................................................................................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
4-20 Comparison Zip1 protein expression after 24 h treatment of 5.0 µM TPEN or
80 µ M zinc. .............................................................................................................25
4-21 Effect of 50 ng/ml of IL1β 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 β treated Caco-2 cells ..................................................29
vii
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
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 µM, zinc sulfate at 40 or 80 µM, IL1 β, 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 β 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
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
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
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).
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
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
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 β 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: 37º 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 µM. Since TPEN has a 1:1 binding capacity with
zinc, three different TPEN concentrations were used: 1.25, 2.5 and 5.0 µM, in order to
mimic a moderate to severe zinc deficiency. Zinc sulfate was used to supplement the
medium with excess zinc (40 and 80 µM). 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
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 –80 oC 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 oC 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 µl) 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 4- to 5-
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
10
chromatography. The methods are comparable to those reported by McMahon and
Cousins (4).
Western Analysis
Equal amounts (30 µg) 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, 1020 µg/ml of total IgG or 2-4 µg/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 4oC at
which time the fixative was changed to a 0.1 M cacodylate buffer. Cells were stored in
this buffer at 4oC until further processing.
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 ½ membrane was placed into fresh LR White and then kept in a 60oC 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 8090 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
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.
Transporter Peptide
m/rZnT1
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
mZIP4
AEETPELLNPETRRL
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 µM Zn than 40 µM 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 µM 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 µM 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 µM TPEN (Figs 45 & 4-6). However, the 48 h time point with 5.0 µM 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 µM 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 µM TPEN) had no effect
13
14
on ZnT7 expression, while 2.5 µM TPEN increased expression 2-fold at 12 h and 48 h
with 2.5 µM TPEN and 5.0 µM TPEN decreased transcript levels 3-fold after 24 h (Fig 410).
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 µM 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 IL1β, 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 Zn- shows that zinc deprivation increased Zip1
expression while zinc excess decreased protein expression by the 24 h time point (Fig 4-
15
20). In addition, treatment with IL1 β 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 β, Zip1 in upregulated, which is in agreement
with mRNA and western data.
16
mRNA Expression Realative to
Control
ZnT-1 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course
10
ZnSO4
40 uM
80 uM
1
0.1
4
8
12
24
48
Tim e (hrs)
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 Course
Relative mRNA Levels
10
TPEN (uM)
1
[1.25]
[2.5]
[5.0]
0.1
0.01
4
8
12
24
48
Hours
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).
17
mRNA Expression Realative to
[0] Control
ZnT-4 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course
10
ZnSO4
40 uM
80 uM
1
0.1
4
8
12
24
48
Tim e (hrs)
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 Course
Relative mRNA Levels
10
TPEN (uM)
1
[1.25]
[2.5]
[5.0]
0.1
0.01
4
8
12
24
48
Hours
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).
18
mRNA Expression Realative to
Control
ZnT-5 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course
10
ZnSO4
40 uM
80 uM
1
0.1
4
8
12
24
48
Tim e (hrs)
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 Course
Relative mRNA Levels
10
TPEN (uM)
[1.25]
[2.5]
1
[5.0]
0.1
4
8
12
24
48
Hours
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).
19
mRNA Expression Realative to
Control
ZnT-6 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course
10
ZnSO4
40 uM
80 uM
1
0.1
4
8
12
24
48
Tim e (hrs)
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 Course
Relative mRNA Levels
10
TPEN (uM)
[1.25]
1
[2.5]
[5.0]
0.1
4
8
12
24
48
Hours
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).
20
mRNA Expression Realative to
Control
ZnT-7 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course
10
ZnSO4
40 uM
80 uM
1
0.1
4
8
12
24
48
Tim e (hrs)
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 Course
Relative mRNA Levels
10
TPEN (uM)
[1.25]
1
[2.5]
[5.0]
0.1
4
8
12
24
48
Hours
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).
21
mRNA Expression Realative to
[0] Control
ZiP-1 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course
10
ZnSO4
40 uM
80 uM
1
0.1
4
8
12
24
48
Tim e (hrs)
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 Course
Relative mRNA Levels
10
TPEN (uM)
[1.25]
1
[2.5]
[5.0]
0.1
4
8
12
24
48
Hours
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).
22
mRNA Abundance Realative to
[0] Control
ZiP-3 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course
10
ZnSO4
40 uM
80 uM
1
0.1
4
8
12
24
48
Tim e (hrs)
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 Course
Relative mRNA Levels
10
TPEN (uM)
[1.25]
1
[2.5]
[5.0]
0.1
4
8
12
24
48
Hours
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).
23
mRNA Expression Realative to
Control
ZiP-4 mRNA Expression in Caco-2 Cells:
Zn Supplementation Time Course
10
ZnSO4
40 uM
80 uM
1
0.1
4
8
12
24
48
Tim e (hrs)
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 Course
Relative mRNA Levels
10
TPEN (uM)
[1.25]
1
[2.5]
[5.0]
0.1
4
8
12
24
48
Hours
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).
24
Caco 2 Ce lls hZiP-1 mRNA IL-B Tre atme nt
7
Fold Change hZiP-1 mRNA
6
5
4
Control
IL-1 B
3
2
1
0
Figure 4-17. Zip1 mRNA expression in Caco-2 cells after 72 hr treatment with 50 ng/ml
ILl β.
Relative abundance of mRNA
ZnT2 mRNA Expression after 72 hr Treatment with
Calcitriol
1.2
1
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=1). Further
testing would be appropriate to confirm these results)
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:
3
Relative Densotometric Units
*
2.5
2
1.5
1
0.5
0
5uM TPEN
80uM Zn
Treatment
Figure 4-20. Comparison Zip1 protein expression after 24 h treatment of 5.0 µM TPEN
or 80 µ M zinc (n=3).
26
Zip1 Protein IL1 B Exp.
5
*
Relative Densitometric units
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Control
IL1 B
Figure 4-21. Effect of 50 ng/ml of IL1β on Zip1 protein after treatment for 24 h (n=3).
27
ZnT2 Protein: Calcitriol treatment
1.4
Relative Densotometric Units
1.2
1
*
0.8
0.6
0.4
0.2
0
Control
Calcitriol
Figure 4-22. Effect of 10 ng/ml of calcitriol on ZnT2 protein expression (n=3).
28
A.
C..
B..
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.
29
A.
B.
C.
D.
Figure 4-25. Electron micrograph of IL1 β 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 µM), 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
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 µM 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 µM 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 µM 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 transporter’s 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 Caco2 cells. This information somewhat agrees with data that Wang et al. (43) found in
32
transfected HEK 293 cells showing that under Zn- conditions, 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 β and vitamin D treatments, respectively. Il1 β 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
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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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.
38