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University of North Carolina at Greensboro, Dept. of Nutrition and Foodservice Management, Greensboro, NC 27402
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • Copper • human T cell • interleukin-2 • gene expression • transcriptional regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover, data also indicate
that even marginally low Cu status influences the development and/or
effector functions of various immune cells (Hopkins and Failla 1995
). However, our current understanding regarding the
biochemical and molecular roles of Cu in the host defense system that
contribute to the maintenance of optimal function remains largely
speculative.
The cytokine interleukin-2
(IL-2)5
is secreted by activated T lymphocytes and has a central role in the
regulation of host reponses to pathogenic challenges. Copper deficiency
attenuates the synthesis of IL-2 by activated rodent splenocytes
(Bala and Failla 1992
), human peripheral blood
mononuclear cells and the human T lymphocyte cell line Jurkat
(Hopkins and Failla 1997
). Because the regulation of
IL-2 gene expression is relatively well understood at the molecular
level, we believe that investigation of the influence of Cu status on
the expression of this gene has the potential to provide insights about
the role(s) that Cu may have in the activities of immune cells.
Cu deficiency decreases both the level of IL-2 bioactivity in medium
and cellular IL-2 mRNA in cultures of activated Jurkat T-cells
(Hopkins and Failla 1997
). Because regulation of IL-2
gene expression at both transcriptional and post transcriptional levels
is involved in tightly controlling the synthesis of this cytokine by
activated T-cells (Crabtree and Clipstone 1994
,
Malter 1998
), low Cu status may decrease IL-2 mRNA
levels by influencing transcription of the IL-2 gene, the stability of
IL-2 mRNA or both processes. IL-2 mRNA is not detected in resting
T-cells. Appropriate stimulation of the T-cell receptor (TcR) and CD28
cell surface receptors (or the use of compounds that mimic stimulation
of these receptors) activates transcription of the IL-2 gene
(Rudd 1996
). The rate of transcription increases rapidly
for several hours and then slowly declines, even in the continued
presence of the stimulators (Jain et al. 1995
). IL-2
mRNA accumulates rapidly for several hours in parallel with the
increased level of transcription. However, the level of IL-2 mRNA
declines more rapidly than can be accounted for by the decreased rate
of transcription, indicating that the stability of IL-2 mRNA is also
actively regulated to modulate the level of mRNA available for
translation (Jain et al. 1995
). Dual regulation of the
rate of both transcription and mRNA degradation provides the cell with
effective mechanisms for the rapid, yet transient, production of IL-2
in response to activation signals.
We have examined the influence of Cu status on transcription of the
IL-2 gene using Jurkat cells stably transfected with a luciferase
reporter gene driven by the 300 bp IL-2 promoter/enhancer region of the
human IL-2 gene. Because the reporter construct does not contain the 3'
untranslated region of the IL-2 mRNA that is believed to mediate the
rapid degradation of the endogenous message (Malter 1998
), the luciferase mRNA is subject only to those factors
that influence the promoter/enhancer region of the endogenous IL-2
gene. Therefore, the stability of IL-2 mRNA in control and Cu deficient
cells has been evaluated separately in the parental (non-transfected)
Jurkat cell line by quantifying the level of mRNA at various times
after treatment of activated cells with a transcription inhibitor. The
results suggest that the reduced level of IL-2 mRNA in Cu deficient,
activated Jurkat cells is due to attenuated transcription of the IL-2
gene and not to alterations in the regulation of the stability of the
mRNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Jurkat and CTLL-2 cells were maintained as described previously
(Hopkins and Failla 1997
) except that 2-mercaptoethanol
was deleted from the complete Roswell Park Memorial Institute medium
1640 (RPMIc). Cu deficiency was induced by incubating cultures in
medium containing 20 µmol 2,3,2-tetraamine/L, a high affinity
chelator of Cu (Fawcett et al. 1980
), for 35 h
before activating cells with phytohemagglutinin-P (PHA-P) and phorbol
myristate acetate (PMA). Details have been described elsewhere
(Hopkins and Failla 1997
).
Reporter gene plasmid.
A plasmid containing the IL-2-luc reporter gene construct, a generous
gift from Dr. Gerald Crabtree (Beckman Center for Molecular and Genetic
Medicine, Howard Hughes Institute, Stanford University), was described
in detail previously (Aoki et al. 1997
, Northrup et al. 1993
). Briefly, this plasmid contains a luciferase cDNA
(deWet et al. 1987
) regulated by the entire IL-2
promoter/enhancer sequence (-326 to +l45), a neomycin resistance gene
under the control of the constituitively active SV-40 promoter and an
ampicillin resistance gene for selection in mammalian and bacterial
cells, respectively. Using plasmid purification kits (Qiagen,Valencia,
CA), plasmid DNA was prepared following the recommended protocols. To
confirm the identity of the plasmid, restriction analysis of plasmid
DNA with endonucleases was performed using conventional methods.
Stable transfection.
Jurkat cells were transfected using DMRIE-C (Life Technologies, Rockville, MD) following the protocol provided by the company. Once conditions for optimal transfection efficiency were determined for this particular cell type and plasmid in a series of transient transfection experiments, 5 x 106 replicating cells were transfected with 10 µg plasmid DNA and 15 µL DMRIE-C in 3 mL RPMI 1640 (no serum, no antibiotic) in the bottom of an upright T75 flask. After 4 h, 20 mL growth medium (RPMIc without antibiotic or fungizone) was added to the flask, and cells were returned to the incubator for recovery and replication. Two days later cells were collected by centrifugation (5 min at 300 x g) and transferred to RPMIG418 (complete RPMI in which penicillin-streptomycin had been replaced by 1.0 g G418/L; Calbiochem, San Diego, CA) for selection of stably transfected cells. Cells were subsequently pelleted and transferred to fresh RPMIG418 every 3–4 d. After culturing in selection media for 9–11 d, a cluster of two to four viable cells was observed per five to 10 microscopic fields. Because cellular debris could not be removed by centrifugation without loss of viable cells, debris was removed slowly from cultures by dilution, i.e., cell cultures were split 1:3 into fresh RPMIG418 every 3–4 d. Because cells from the original transfections were divided into multiple flasks at the initiation of selection, 20 stably transfected, multiclonal cell lines were generated. These cell lines were cultured in selection medium for a month as described above until cellular debris was no longer evident and cell populations were sufficient to evaluate luciferase activity. The multiclonal cell lines were designated Jurkat/IL-2 Luciferase multiclonal cell lines 1–20 and will be referred to below as J/IL-2L1, J/IL-2L2, etc.
Clonal cell lines (i.e., a population originating from a single cell) were generated by limiting dilution from one of the multiclonal cell lines that exhibited the highest luciferase activity (viz., J/IL-2L5). These clones were grown in selection medium for 4–5 wk before the level of luciferase activity in those that exhibited robust growth was analyzed. Only 25% of the cloned cell lines that grew in selection medium contained detectable luciferase activity. The luciferase containing clonal cell lines are designated below as J/IL-2L5.1, J/IL-2L5.2, etc.
Luciferase assay.
Luciferase activity in transfected cells was evaluated using reagents from The Luciferase Assay System with Reporter Lysis Buffer (Promega, Madison, WI) following the recommended protocol with minor modifications. Briefly, cells were incubated with or without 2,3,2-tet for 35 h, collected, counted and reseeded into 24-well plates at 1 x 106 cells per well in 1 mL fresh RPMIG418containing 2 mg PHA-P/L and 10 µg PMA/L. Cells were collected 20 h after activation and pelleted (300 x g for 10 min at 4°C). Supernatants were removed and stored at -70°C for analysis of IL-2 bioactivity. One hundred µL of 1 X Reporter Lysis Buffer was added to each cell pellet without washing the pellets. Cells were repipetted five times to ensure lysis of all cells and lysates were stored at -70°C. For analysis, lysates were thawed on ice, duplicate or triplicate aliquots (20 µL) were equilibrated to room temperature and mixed with Luciferase Assay Reagent (50 µL), and light output was quantified for 30 or 60 s in a Lumat LB 9501 luminometer (Berthold Systems, Pittsburgh, PA). Light output in samples prepared from non-transfected cells was similar to that in sample blanks containing only Luciferase Assay Reagent. Control samples (transfected cells that had not been activated) contained minimal levels of luciferase activity that varied proportionately with the level of luciferase activity in activated cultures.
Interleukin-2 bioactivity.
IL-2 activity in culture supernatants was determined as described
previously (Hopkins and Failla 1995
).
mRNA stability.
Stability of IL-2 mRNA in control and Cu-deficient parental Jurkat
cells was evaluated by conventional methods (Current Protocols 1991
).
Briefly, control and chelator-treated cultures of Jurkat cells were
stimulated with 2 mg PHA-P/L and 10 µg PMA/L.
5,6-dichlorobenzimidazole riboside (DRB; 20 mg/L; cat. #D-1916, Sigma
Chemical, St. Louis, MO) was added to cultures 6 h after
activation to inhibit transcription. Previous studies had revealed that
IL-2 mRNA in Jurkat cells was maximal 6 h post activation
(Hopkins and Failla 1997
). Total RNA was isolated at
indicated times, and the levels of IL-2 mRNA and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were evaluated by
Northern blot analysis. cDNA probes for IL-2 and GAPDH were generated
by polymerase chain reaction (PCR) using primers and protocols as
described previously (Hopkins and Failla 1997
).
Statistical analysis.
Data were analyzed by Student's t-test or ANOVA using the
General Linear Models (GLM) procedure followed by Tukey's multiple
range test (SAS Institute, Cary, NC) when indicated. Results are
presented as means ± SEM (n = 3) for
representative experiments. Differences are considered significant when
P 
0.05. All experiments were repeated at least
twice. Although actual values for control and experimental samples
often varied between experiments, significant differences in values in
response to changes in Cu status (i.e., 2,3,2-tet treatment) were
similar in all replicate experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Initially, the level of
luciferase activity in 20 multiclonal cell lines was evaluated.
Expression varied widely among the different cell lines as shown in
Figure 1
. Luciferase activity in cells activated with PHA and PMA ranged from a
low of 16,000 relative light units (RLU)/2 x 105
cells to a high of 300,000 RLU/2 x 105 cells.
Nonactivated cells contained minimal levels of luciferase activity (0.4
RLU/2 x 105 cells to 3,500 RLU/2 x
105 cells) that varied proportionately with the level of
luciferase activity in activated cultures. Thus, luciferase expression
increased 15- to 80-fold after stimulation with PHA/PMA in all cell
lines.
|
). Luciferase activity was 56, 53 and 32% lower in
J/IL-2L5, J/IL-2L14 and J/IL-2L15 cells, respectively, compared to the
cultures that were not exposed to chelator (Fig. 2
). Moreover, the inhibitory effect of pretreatment with the Cu chelator
on reporter gene activity in activated J/IL-25 cells was
dose-dependent, ranging from 10 to 60% for cultures treated with 5–40
µmol/L 2,3,2-tet (Fig. 3
). The relative levels of IL-2 bioactivity secreted into the supernatants
of these same cultures was similarly reduced in a dose-dependent manner
(Fig. 3)
and was highly correlated (r = 0.99) with
luciferase activity in the cells. These data suggested that cellular Cu
deficiency attenuates IL-2 synthesis in Jurkat cells primarily by
inhibiting transcription from the IL-2 promoter/enhancer.
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).
|
.
Moreover, the level of reporter activity decreased by 35–45% in three
of the multiclonal lines over a period of several months of culturing
in RPMIG418 (data not shown). Therefore, a parallel series
of studies were performed with clonally derived populations of stably
transfected Jurkat cells. A dozen single clone cell lines that grew
robustly in medium containing G418 were generated by limiting dilution
from the multiclonal line J/IL-2L5. Four of these clones contained
detectable luciferase activity in PHA/PMA stimulated cells. However,
the level of luciferase activity in these clonal cell lines was
markedly lower than that in the multiclonal parental cell line (about
60,000 RLU/2 x 105 cells compared to 250,000
RLU/2 x 105 cells, respectively). The influence of Cu
status on transcriptional activity of the luciferase reporter was
examined in two of these clones in experiments similar to those
described above. Similar to the responses of the multiclonal cell
lines, chelator-induced Cu deficiency significantly (P <
0.05) reduced luciferase activity in PHA/PMA activated clonal cell
lines (Fig. 5
), and the addition of Cu, but not Zn or Fe, blocked the reduction in
luciferase activity (data not shown).
|
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). IL-2 gene
expression, like that of many other immune response and growth
regulatory genes, is regulated by both transcriptional and
post-transcriptional mechanisms (Malter 1998
). This dual
regulation provides cells with the capacity to rapidly initiate and
upregulate, as well as quickly terminate, gene expression in response
to appropriate signals. Transient production of cytokines is consistent
with their roles as immunoregulatory factors. A rapid host response to
pathogenic challenge is essential for controlling and eliminating
pathogens. On the other hand, the intensity and duration of immune
system activation must be strictly controlled to limit the deleterious
effects of immune activities, e.g., tissue damage caused by
inflammatory processes.
Transcription of the IL-2 gene is regulated by the complex interaction
of four known transcription factors within the 300 bp promoter/enhancer
region immediately 5' of the coding region (Garrity et al. 1994
, Jain et al. 1995
, Weiss and Littman 1994
). In comparison to the regulation of IL-2 gene
transcription, regulation of the stability of IL-2 mRNA is still
relatively poorly understood. The turnover of messenger RNA for IL-2
and a number of other lymphokines, cytokines and oncogenes appears to
be regulated at least in part by UUAUUUA (U/A) motifs found in the
3'-untranslated region (3'-UTR) of the mRNAs for these genes
(Malter 1998
). This nonamer is essential for the rapid
degradation of IL-2 mRNA in nonstimulated T cells. The half-life of
IL-2 mRNA is markedly increased in activated lymphocytes, although the
cis element(s) that mediate this increased stability
remain(s) unknown (Chen et al. 1998
, Malter 1998
).
It seemed likely that Cu status could alter IL-2 mRNA levels by
influencing the rate of IL-2 gene transcription and/or the stability of
IL-2 mRNA. We elected to examine transcriptional activity of the IL-2
gene by using a luciferase reporter construct driven by the 300 bp
human IL-2 promoter/enhancer. We decided that stably transfected cell
lines would be more appropriate than transient transfection because the
experimental paradigm used to examine the effect of Cu deficiency on
activated Jurkat cells requires incubation of the cultures with
2,3,2-tet for 35 h followed by activation of cells with PHA and
PMA for an additional 20 h before collecting the medium and cells
for assessment of IL-2 bioactivity and luciferase activity,
respectively. Exogenous DNA has a limited lifetime in transiently
transfected cells because nucleases and cell division degrade and
dilute the foreign DNA (Alam and Cook 1990
).
Consequently, the transcriptional activity of a reporter gene usually
peaks within 1–3 d after transfection and subsequently declines and is
undetectable after a week. Alam and Cook (1990)
recommend stimulation of an inducible reporter gene within 24 h of
transfection because induction after peak transcriptional activity
tends to underestimate the level of transcription of the test DNA and
may even falsely indicate that the regulatory sequence is inactive.
Therefore, stably transfected multiclonal cell lines containing the
IL-2 promoter/enhancer driven luciferase construct were generated.
Luciferase activity in lysates of PHA/PMA-activated cells from Cu
deficient cultures of three different multiclonal cell lines was
35–55% less than that of cells that had not been exposed to 2,3,2-tet
(Fig. 2)
.
In stably transfected cells, integration of the exogenous DNA occurs
randomly and expression of the integrated DNA is influenced by the
surrounding chromosomal environment. Moreover, multiple copies of the
construct are frequently incorporated into the genome, and the
exogenous DNA may also be fragmented prior to integration. Such random
integration likely contributed to the variability in the level of
luciferase activity detected in the different multiclonal cell lines.
Moreover, we observed that the level of luciferase activity in our
multiclonal cell lines declined over several months time in culture.
Similarly, de Wet et al. (1987)
reported that expression
of a reporter construct in a multiclonal cell line tended to decline
slowly over time (50% over 25 generations), even in the continued
presence of G418. Therefore, several clonal cell lines were generated,
and the impact of Cu deficiency on the activity of the reporter gene in
these cell lines was also examined. The results were similar to those
observed in the multiclonal lines, i.e., low cellular Cu status
decreased the level of luciferase activity in the lysates of activated
cells. The observation that Cu deficiency reduced the expression of the
reporter gene in several, separate, multiclonal and clonal cell lines
indicates that the IL-2 promoter/enhancer itself, rather than the local
environment of the reporter construct, is influenced by the decreased
availability of Cu.
The addition of a slight molar excess of Cu, but not Zn or Fe, to
medium containing 2,3,2-tet prevented the chelator-mediated decline in
luciferase expression in activated J/IL-2L5 cells (Fig. 4)
. This
suggests that the reduction in IL-2 gene transcription is specifically
mediated by decreased Cu status of the cells and not a secondary iron
deficiency. This conclusion is supported by our previous finding that
exposure of the parental line of Jurkat cells to 2,3,2-tet did not
increase the level of cell surface transferrin receptors
(Hopkins and Failla 1997
), a well established response
to cellular Fe deficiency (Leibold and Guo 1992
).
The activity or abundance of reporter gene products generally is
directly proportional to the transcriptional activity of the regulatory
sequences inserted into their vectors (Alam and Cook 1990
). Although a reporter gene is an indirect measure of
transcriptional activity, it has the advantage of providing assessment
of that activity within the intact in vivo environment. Therefore, the
data suggested that Cu deficiency attenuates IL-2 synthesis primarily
by inhibiting transcription from the IL-2 promoter/enhancer rather than
by influencing post-transcriptional regulation. To confirm this we
examined the stability of IL-2 mRNA in Cu-deficient Jurkat cells. The
rate of IL-2 mRNA degradation in Jurkat cells was found to be
independent of cellular Cu status. This observation is consistent with
the absence of any reports in the literature suggesting that the
stability of IL-2 mRNA is regulated by nutritional status.
Other investigators have reported that deficiencies of a variety of
nutrients and dietary components other than Cu also attenuate the
production of IL-2; these include fatty acids (viz., arachidonic,
eicosopentaenoic and docosahexaenoic acids), zinc, iron and vitamins E,
B-6 and D (Munoz et al. 1995
). With the exception of
vitamin D, the potential impact of these nutrients on the
transcriptional regulation of the IL-2 gene has not been examined.
Alroy et al. (1995)
showed that the active metabolite of
vitamin D, 1,25-dihydroxycholecalciferol, inhibited transcription of
the IL-2 gene by blocking NF-ATp/AP-1 complex formation at the distal
NFAT response element of the IL-2 promoter/enhancer.
Cu was shown to directly control the expression of specific genes in
bacteria and simple eukaryotes. For example, Cu binds to and regulates
the activity of ACE1 and AMT1, transcription factors that recognize
response elements in the promoter regions of metallothionein genes in
yeast (Thiele 1992
). There are no known examples of Cu
directly regulating gene expression in mammalian cells. However,
Wilson et al. (1997)
showed that Cu deficiency is
associated with increased transcription of the fatty acid synthase gene
in the rat liver. This increased transcriptional activity is apparently
caused by altered hepatic thiol status, i.e., an increased level of
reduced glutathione (GSH) and an increased ratio of reduced to oxidized
glutathione (GSH:GSSG). Examination of the possibility that Cu status
modulates the transcriptional efficiency of the IL-2 gene by altering
the cellular redox environment in lymphocytes merits consideration.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Presented in abstract form at Experimental Biology
98, April 1998, San Francisco, CA [Hopkins, R. G and Failla, M. L. (1998) Copper deficiency inhibits transcription from the
interleukin-2 promoter in stably transfected Jurkat cells. FASEB 12:
A200 (abs.)].
2 Supported in part by the North Carolina
Agricultural Research Station.
3 The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance with 18
USC section 1734 solely to indicate this fact.
5 Abbreviations used: 2,3,2-tet, 2,3,2-tetraamine;
3'-UTR, 3'-untranslated region; Cu-Zn SOD, Cu, Zn superoxide dismutase;
DRB, 5,6-dichlorobenzimidazole riboside; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GLM, General Linear Models;
GSH, reduced glutathione; IL-2, interleukin-2; PCR, polymerase chain
reaction; PHA-P, phytohemagglutinin-P; PMA, phorbol myristate acetate;
RLU, relative light units; RPMI 1640, Roswell Park Memorial Institute
1640 medium; TcR, T-cell receptor; U/A, UUAUUUA.
Manuscript received August 5, 1998. Initial review completed September 29, 1998. Revision accepted November 13, 1998.
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