Apolipoprotein A-I gene expression is regulated by cellular
zinc status in Hep G2 cells
John Y. J.
Wu,
Yan
Wu,
Scott K.
Reaves,
Yi Ran
Wang,
Polin P.
Lei, and
Kai Y.
Lei
Department of Nutritional Sciences, University of Arizona, Tucson,
Arizona 85721
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ABSTRACT |
The influence of Zn on the expression of the apolipoprotein A-I
(apoA-I) gene in Hep G2 cells was examined. Zn depletion was achieved
with a low-Zn (ZD) medium prepared from Zn-free growth medium
(Opti), a ZD medium containing Chelex 100-extracted fetal bovine serum (CHE), and a medium containing chelator
1,10-phenanthroline (OP). Compared with those for their respective
controls, cellular Zn levels were reduced by 55, 48, and 46% and
apoA-I mRNA abundances were reduced by 20, 29, and 28% in Opti, CHE,
and OP systems, respectively, after one passage in ZD media or 24 h in
OP medium. To establish the specificity of Zn treatment, groups of ZD
cells were treated with their respective control media for the last 24 h (ZDA) or normal cells were cultured with OP medium supplemented with
Zn (OP-Zn). ZDA treatments partially normalized cellular Zn levels in
the Opti system and restored or elevated apoA-I mRNA levels in the Opti
or CHE system, respectively. Similarly, the OP-Zn treatment restored
the cellular Zn and apoA-I mRNA levels. Furthermore, one passage of
culture with Zn-supplemented media in both the Opti and CHE systems
resulted in higher cellular Zn and apoA-I mRNA levels than those for
controls. Most significantly, short-term high-Zn induction to normal
cells markedly elevated the cellular Zn (3-fold) and apoA-I mRNA
(5-fold) levels. Data derived from this study strongly suggest that the
expression of apoA-I is regulated by cellular Zn status.
cardiovascular disease; cholesterol; high-density lipoprotein
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INTRODUCTION |
THE HYPOCHOLESTEROLEMIC EFFECT of Zn deficiency, mainly
the reduction of high-density lipoprotein (HDL) cholesterol, in animals (3, 9, 11) and humans (8, 14) has been documented. When
the HDL fraction was examined by compositional and chromatographic analyses, Zn deficiency significantly reduced the total amount of
plasma HDL particles, with no influence on the percent composition of
total protein, triglycerides, phospholipids, and cholesterol (9). The
reduction in the HDL cholesterol was mainly due to a marked decrease in
apolipoprotein E (apoE)-free HDL, the major subclass of the HDL
fraction (10). No alteration in very-low-density lipoprotein and
low-density lipoprotein cholesterol levels was produced by Zn depletion.
In view of the association of low plasma HDL cholesterol and apoA-I
levels with increased risk of atherosclerosis, the changes in
lipoprotein metabolism induced by Zn deficiency may in part contribute
to the development of atherosclerosis. Dietary Zn deficiency was found
to be able to decrease the plasma apoA-I levels in both rats and
hamsters (18). In addition, the hepatic apoA-I and metallothionein-II
(MT-II) mRNA abundances were similarly altered by the Zn status. To
further establish whether the observed alterations were truly due to Zn
deficiency, groups of Zn-deficient (ZD) animals were given a
Zn-adequate diet for 2 days. Such Zn replenishment either raised the
plasma apoA-I level, as well as the hepatic MT-II and apoA-I mRNA
abundances, to levels higher than control levels in rats or normalized
them to the control levels in hamsters (18). These observations
strongly suggested that Zn deficiency could specifically downregulate
the hepatic expression of the apoA-I gene at a pretranslation step. The
establishment of a suitable in vitro Zn deficiency model will
facilitate the further investigation of the mechanism(s) responsible
for such regulation.
In the present study, we have established three systems to manipulate
the cellular Zn level in cultured Hep G2 cells, which could be used as
in vitro models to study the influence of Zn status on human hepatic
gene expression. In the first two systems, Hep G2 cells were cultured
in two different low-Zn media to deplete cellular Zn over one passage,
whereas in the third system, Zn depletion was achieved by exposing
nearly confluent cells to a Zn chelator for 24 h. Furthermore, the
treatment specificity was established for each system by the repletion
of Zn. Moreover, long-term moderately high Zn treatment and short-term
high-Zn treatment were also used as the Zn supplementation treatments. In all these systems, the cellular Zn levels and MT-II mRNA abundances were modulated by various treatments. Consistent with the results from
in vivo studies, data derived from this study strongly suggested that
Zn status may directly affect apoA-I gene expression.
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MATERIALS AND METHODS |
Cell culture and treatment.
The human hepatoblastoma cell line Hep G2 was obtained from the
American Type Culture Collection (Manassas, VA) and was used to mimic
the human hepatic responses to different zinc statuses. All reagents
were obtained from Life Technologies (Grand Island, NY). Cells were
maintained in a regular medium composed of 90% DMEM-10%
heat-inactivated fetal bovine serum (FBS), 1% nonessential amino
acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml
streptomycin. Medium was replaced twice a week, and 7 days of culture were used as one passage. Nearly confluent cells at the end
of passage 80 were subcultured at the
ratio of 1:8 from a T75 flask to a 100-mm tissue culture dish and used
as experimental cells.
Three different treatments were used to deplete cellular Zn from Hep G2
cells. In the first two experiments, Zn depletion was achieved by
culturing cells in low-Zn media for one passage. The Opti-MEM contains
supplemented growth factors, and it is designed as a serum-free or
serum-reduced medium for a DNA transfection assay (Life Technologies).
A customized Zn-free Opti-MEM formulation was obtained from Life
Technologies. In a preliminary study, Hep G2 cells were cultured for
one passage in such medium, with 1% FBS and different levels of Zn
addition (0 to 16 µM). The cellular Zn levels were altered in a
dose-dependent manner (data not shown). In addition, no apparent
morphological alterations could be observed within one passage of
culture (data not shown). For the experiment, the ZD medium was
prepared by adding 1% FBS to Zn-free Opti-MEM and was found to contain
0.4 µM Zn. The Zn-adequate (ZA4) medium was prepared by adding
ZnSO4 to the ZD medium to the
level of 4 µM Zn (which was equal to the Zn level found in
regular Hep G2 culture media with 10% FBS). The Zn-supplemented (ZA16)
medium was prepared by adding Zn to ZD medium to the level of 16 µM
Zn (which was equal to the Zn level found in the human plasma). Cells were cultured in normal medium for the first 12 h and then switched to
ZD, ZA4, or ZA16 medium for the rest of the passage. A group of ZD
cells were replenished for the last 24 h with the ZA4 medium and used
as the ZDA cells. Four plates from each group were used for RNA isolation, and another four plates were used for the
measurement of cellular Zn level.
In the second experiment, a divalent ion-chelating resin was used to
remove Zn from FBS. Chelex 100 resin (Bio-Rad, Hercules, CA) was mixed
with FBS at a 1:4 ratio (wt/vol), and the suspension was shaken for 2 h
at 4°C, as described previously (5, 12). Chelex 100 resin was
separated from FBS by centrifugation, and the extracted serum was
further passed through a 0.4-µm filter for sterilizing serum and
removing any remaining Chelex 100 resin. The resultant serum only
contained the background level of Zn, as detected by atomic absorption,
and the medium containing 90% DMEM and 10% such serum was considered
Zn-free medium (basal medium). When cells were cultured directly in
this basal medium, they could not reach levels of confluency that were
the same as that for cells cultured in regular medium (data not shown),
suggesting that cell growth was affected by the low-Zn status of the
basal medium. The minimal supporting level of Zn was determined by a dosage curve, with the DNA content per plate as the index for the cell
growth. At the low end (0.1-0.4 µM), the supplementation of Zn
resulted in dosage-dependent increases in DNA content per plate (Fig.
1A;
for clear illustration, only the data representing 0.2 and 0.4 µM are
shown). However, the higher dosage of Zn supplementation resulted in
essentially the same level of DNA content, which was comparable to that
observed in cells cultured in the normal medium containing regular FBS
(Fig. 1A). In contrast, the
cellular Zn content exhibited continuous dosage-dependent increases
over the entire testing range of Zn supplementation (Fig.
1B). These observations suggested
that the basal medium containing 0.4 µM Zn could support the normal
level of cell growth and maintain a depleted cellular Zn status. For
the experiment, the ZD medium was the basal medium supplemented with
0.4 µM Zn. Similarly, the ZA4 medium and the ZA16 medium were
prepared by adding 4 and 16 µM Zn to the basal medium, respectively,
to mimic the Zn level observed in normal culture medium or in human
plasma. Hep G2 cells were cultured either in ZD, ZA4, or ZA16 medium
for one passage. As for the first experiment, a group of ZDA cells was
also included. Four plates for each group were used for RNA isolation,
and another four plates were used for the measurements of cellular Zn
and DNA contents.

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Fig. 1.
Determination of optimal level of Zn in medium for depletion of
cellular Zn from Hep G2 cells. Basal medium was prepared with 90%
DMEM-10% Chelex 100-treated serum. Cells were cultured in basal medium
supplemented with graded levels of Zn (only 0.2, 0.4, 1, and 4 µM
levels are shown) for one passage. Another group of cells was cultured
in normal medium and used as control. Cellular Zn and DNA contents were
measured by atomic absorption spectrophotometry and diphenylamine
procedure, respectively. Values are means ± SE from 4 experiments.
Means with different letters are significantly different
(P < 0.05 by 1-way ANOVA).
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1,10-Phenanthroline (OP; Sigma, St. Louis, MO) is a Zn chelator widely
used to remove Zn in cell-free systems, as well as to deplete cellular
Zn from various types of cells (1, 7). In the third experiment of this
study, OP was used to deplete cellular Zn from Hep G2 cells. Cells were
cultured for 6 days in normal medium, and then 1% (vol/vol) of
100× OP stock, prepared in ethanol, was added to the culture
medium. The minimal effective dose of OP was determined by dosage
curves. As depicted in Fig. 2A, cells
exposed to graded levels of OP for 24 h experienced dose-dependent
reductions of cellular Zn contents. However, higher doses
of OP (300 and 400 µM) may also affect the cellular metabolism, as
evidenced by the significant decreases of DNA content per culture plate. Therefore, 200 µM was selected as the suitable dose and used
for further evaluation of the optimal treatment time. In the time
course assay, the Zn depletion effect of 200 µM OP was not exhibited
until 18 h of treatment, and it was not further enhanced by extending
the treatment to 36 h (data not shown). Thus 24 h appeared to be the
optimal treatment duration. To examine whether any possible changes
caused by the OP treatment are specifically due to the low-Zn status or
other possible side effects of OP, increasing amounts of Zn were added
back to OP-treated cells to counteract OP's Zn depletion action. As
depicted in Fig. 2B, Zn replenishment
resulted in dose-dependent increases in cellular Zn levels. Zn at 40 µM was found to be able to cause the reversion of the cellular Zn
level to that of the controls. Although the cellular DNA content in
cells treated with 40 µM Zn was slightly higher than that in
unreplenished OP-treated cells, it was found not to be significantly
different from that in the untreated control cells. The OP medium was
prepared by adding 1% (vol/vol) 100× OP stock in ethanol to the
regular culture medium. The control medium was prepared by adding the
same amount of ethanol (OP carrier) to the regular medium, and the
Zn-supplemented (OP-Zn) medium was prepared by adding 40 µM Zn into
the OP medium. Cells were cultured in the normal medium for 6 days and
then exposed to control, OP, or OP-Zn medium for another 24 h. Four
plates were used for DNA and Zn analyses, and another four plates were
used for RNA isolation.

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Fig. 2.
Determination of effective levels of 1,10-phenanthroline (OP) for
depletion of cellular Zn (A) and of
supplemented Zn for reversal of OP effect
(B) in Hep G2 cells. Cells were
cultured in regular medium for 6 days and then treated for another 24 h
with media with increasing amount of OP
(A) or increasing amount of Zn-200
µM OP (B). For controls, cells
were cultured in normal medium, with same amount of ethanol used as OP
carrier in OP medium and in OP medium supplemented with Zn (OP-Zn
medium), for the last 24 h. Values are means ± SE from 4 experiments.
Means with different letters are significantly different
(P < 0.05 by 1-way ANOVA).
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To further examine the effect of Zn status on apoA-I gene expression, a
short-term Zn induction experiment was performed. Cells were cultured
in regular medium for nearly 7 days. In the Zn induction (ZI) group,
cells were then exposed to the regular medium supplemented with 200 µM ZnSO4 for 4 h, whereas the
control cells were continuously cultured in the regular medium. The
cells were then harvested, four plates for each group were used for DNA
and Zn analyses, and another four plates were used for RNA isolation.
Determination of cellular contents of Zn and DNA.
Cells were washed twice with PBS and harvested. After a centrifugation
at 500 g for 5 min at 4°C, the
cell pellet derived from one plate was resuspended in 1.5 ml PBS and
sonicated. One milliliter of this sonicant was directly used to measure
the cellular Zn content by means of a flame atomic absorption
spectrophotometer (Hitachi), against the standard curve of
0.05-1.0 µg/ml of Zn (19). The remaining 0.5 ml of sonicant was
used for the measurement of cellular DNA content (17). Total cellular
contents of Zn per microgram of DNA are presented, because a linear
relationship between the amount of cellular DNA and cell number,
regardless of treatments, was previously established.
RNA isolation and analysis.
In the first experiment (with Opti-MEM), total cellular RNA was
isolated by using the TRIzol reagent (Life Technologies), according to
the manufacturer's instructions. The apoA-I mRNA abundance was then
analyzed by Northern blot analysis, as described previously (19). 18S
rRNA was used as the normalization reference. In all other experiments,
RNA was isolated by using the RNAqueous kit (Ambion, Austin, TX),
according to the manufacturer's instructions. The abundance of apoA-I
mRNA was measured by an RNase protection assay (RPA) with the RPA-II
kit (Ambion). To provide another index for the cellular Zn status and a
positive control for Zn-regulated gene expression, MT-II mRNA abundance
was also measured. The abundance of 18S rRNA was used as the internal
reference for the normalization, and an 18S antisense template was
purchased from Ambion. The RPA probe synthesized by T7 RNA polymerase
was 128 nt in length. The 80-nt protected fragment of 18S rRNA appeared
as double bands in RPA gels.
The human MT-II antisense template was prepared by RT-PCR, as
previously described (15, 18). A pair of human MT primers, MT5
and MT3, corresponding to the 5' and 3' regions,
respectively, of human MT-II cDNA (15), was synthesized. RT-PCR
products were cloned into the pGEM-T PCR cloning vector (Promega).
Plasmid DNA was isolated from a correct clone, which contained an MT-II
cDNA fragment in the antisense orientation with respect to the T7
promoter. A pair of primers (puc/M13F and Rev-T), corresponding
to the upstream and downstream vector sequences, was used to
prepare the final cDNA template for MT-II RPA probe synthesis in a
PCR. The resulting fragment was 354 nt in length, and the
RNA probe transcribed from the T7 promoter was 288 nt in length and
contained 201 nt of human MT-II antisense sequence.
Similarly, the human apoA-I antisense template was prepared from human
apoA-I cDNA, a kind gift from Dr. Lawrence Chan (Baylor College of
Medicine, Houston, TX). A pair of primers, hAI-5 and hAI-3, was used to
amplify a 157-bp region (nt 364-520). The final cDNA template was
310 nt in length, and the RNA probe transcribed from the T7 promoter
was 244 nt in length and contained 157 nt of human apoA-I antisense sequence.
The following primers were synthesized by Life Technologies: MT5,
5'-ATG GAT CCC AAC TGC TCC TGC G-3'; MT3, 5'-AGG GCT
GTC CCA GCA TCA GGC-3'; hAI-5, 5'-AGG AGT TCT GGG ATA ACC
TGG-3'; hAI-3, 5'-GGC TCC ACC TTC TGG CGG TA-3';
pUC/M13F, 5'-CGC CAG GGT TTT CCC AGT CAC GAC-3'; and Rev-T,
5'-TGC AGG CGG CCG CAC TAG TGA TT-3'.
The RPA probes for apoA-I, MT-II, and 18S were synthesized by using the
MAXIscript in vitro transcription system (Ambion), with T7 RNA
polymerase. These probes were labeled at different predetermined
specific activities by adjusting the ratio of
[32P]UTP to cold UTP
in the labeling reaction mixture to provide roughly
similar band intensities in the final RPA gel.
In the RPA experiment, each sample reaction mixture consisted of 5 µg
of total RNA derived from Hep G2 cells after various treatments, 0.05 fmol of human apoA-I and MT-II RPA probes, 400 ng of 18S probe, and 5 µg of yeast RNA. The RNA and probes were coprecipitated and
resuspended in hybridization buffer and then hybridized overnight at
45°C. A diluted RNase cocktail was then added, and the mixture was
further incubated at 37°C for 30 min. The RNase digestion was
inactivated by the addition of inactivation buffer. The protected RNA
probes were precipitated and separated by 6% acrylamide-8 M urea gel.
To establish the specificity for the RPA signals, both a negative
control and a positive control were included in each RPA gel. In the
negative control, 5 µg of sample RNA were replaced with an equal
amount of yeast RNA, and then the control mixture was processed through
the same procedure as that used for the sample reactions.
No protected signal could be detected in the negative control. The
positive control was processed in the same manner as were the sample
reaction mixtures but was not digested by RNase. Only full-length
probes were observed in the positive control. The RPA gels were dried
and autoradiographed. Band intensities of protected signals were
quantified by a laser densitometer (Molecular Dynamics). The relative
mRNA abundances in each sample were expressed as the arbitrary units of
the apoA-I or MT-II band per arbitrary unit of the internal reference
18S in the same RPA reaction.
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RESULTS |
Zn depletion decreased the cellular apoA-I mRNA abundance.
Significant reductions in cellular Zn levels were observed in cells
from the Zn depletion treatment in all three systems. Compared with
those of their respective controls, the cellular Zn level was reduced
55 or 48% by treatment with the ZD medium based on Opti-MEM or Chelex
100-extracted serum, respectively (Table
1). In addition, the OP treatment resulted
in a 46% reduction in cellular Zn level (Table 1).
Consistent with the changes in cellular Zn levels, the cellular MT-II
mRNA abundance was depressed by the Zn depletion
treatments. As shown in Figs. 3 and
4, the MT-II mRNA levels were decreased to 38 and 43% of
their respective controls by either one passage of culture in ZD Chelex
100 medium or 24 h of OP treatment. In Fig. 3, the reduction of MT-II
mRNA abundance appeared not to be significant, primarily because of the
highly elevated MT-II mRNA in ZDA and ZA16 groups. When the ZD- and
ZA4-treated groups were analyzed separately, it was found
that the reduction in MT-II mRNA was significant.


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Fig. 3.
Effect of Zn status of medium on cellular metallothionein-II (MT-II)
mRNA and apolipoprotein A-I (apoA-I) mRNA levels in Hep G2 cells. Cells
were cultured for 1 passage in Zn-deficient (ZD), Zn-adequate (ZA4), or
Zn-supplemented (ZA16) medium, which was prepared by adding Zn as a
supplement to Chelex 100-extracted, serum-based basal medium. The ZDA
cells were ZD cells exposed to ZA4 medium for the last 24 h. MT-II and
apoA-I mRNA abundances were measured by RNase protection assay, with
18S rRNA as reference. Values are means ± SE from 4 experiments.
Means with different letters are significantly different
(P < 0.05 by 1-way
ANOVA).
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Fig. 4.
Effect of OP and OP-Zn treatments on cellular MT-II mRNA and apoA-I
mRNA levels in Hep G2 cells. Cells were cultured in normal medium for 6 days and then treated with control (CT), OP, or OP-Zn medium for last
24 h. MT-II and apoA-I mRNA abundances were measured by RNase
protection assay, with 18S rRNA as reference. Values are means ± SE
from 4 experiments. * Significantly different from control
(P < 0.05 by 1-way
ANOVA).
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In addition to causing changes in the cellular Zn status and MT-II mRNA
abundance, the Zn depletion treatments also significantly depressed
apoA-I mRNA levels by 20 or 29% in the ZD cells cultured in the
Opti-MEM system (Fig. 5) or in Chelex
100-based ZD medium (Fig. 3), respectively. Moreover, OP
treatment also resulted in a 28% reduction in cellular apoA-I mRNA
level (Fig. 4).

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Fig. 5.
Effect of Zn status of media on cellular apoA-I mRNA abundance in Hep
G2 cells. Cells were cultured for one passage in ZD, ZA4, or ZA16
medium, which was prepared by adding Zn as a supplement to
Opti-MEM-based basal medium. ZDA-treated cells were ZD cells exposed to
ZA4 medium for the last 24 h. apoA-I mRNA abundance was measured by
Northern blotting, with 18S rRNA as reference. Values are means ± SE
from 4 experiments. Means with different letters are significantly
different (P < 0.05 by 1-way
ANOVA).
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Alterations in cellular apoA-I mRNA levels appeared to be Zn
specific.
To examine the specificity of the Zn depletion effect on cellular
apoA-I mRNA abundance, groups of ZD cells previously treated with ZD
media were replenished for 1 day with their respective ZA4 control
media. In the Opti-MEM system, such repletion elevated the cellular Zn
level from 45% of control (ZD) to 81% of control in ZDA cells (Table
1). Although this level was still lower than that observed in
ZA4-treated cells, the apoA-I mRNA level was already
normalized to the control level (Fig. 5). Similar results were obtained
from the Chelex 100 system. Compared with results for the unreplenished
ZD-treated cells, ZDA treatment elevated the cellular Zn level from 52 to 81% of the control (Table 1). However, the same treatment
significantly increased the cellular MT-II (448%) and apoA-I (174%)
mRNA levels above those for the ZA4 controls (Fig. 3).
In the OP experiment, the addition of 40 µM Zn to the OP medium at
the beginning of the OP treatment prevented the Zn depletion effect of
OP, and the cellular Zn level was the same as that for the controls
(Table 1). Consistent with the cellular Zn level, the cellular MT-II
and apoA-I mRNA abundances were also not altered in OP-Zn cells (Fig.
4). These observations suggested that the reduction of apoA-I mRNA
abundance in the OP cells did not result from possible side effects of OP.
Zn supplementation increased cellular apoA-I mRNA abundance.
The influence of higher cellular Zn levels on apoA-I gene expression
was also examined in the study. Because the Zn level in human plasma
was found to be ~16 µM, this level was selected as the
physiological level for Zn supplement. In the Opti-MEM cul ture
system, cells cultured in ZA16 medium had a higher (164% of ZA4
control) cellular Zn content (Table 1). The ZA16 treatment also
resulted in a small but significant increase (11%) in the cellular apoA-I mRNA abundance, compared with that for the ZA4 controls
(Fig. 5). Similar results were also observed with the Chelex 100 system. Compared with results for the ZA4 controls, the cellular Zn and
MT-II mRNA levels were elevated to 123 and 540%, respectively (Table
1; Fig. 3). Accompanying the increased Zn level, the apoA-I mRNA
abundance was coordinately increased to 173% of controls by the ZA16
treatment (Fig. 3).
To further test the concept that cellular Zn status may affect apoA-I
gene expression, a short-term Zn induction experiment was performed.
Cells were cultured in normal medium for the entire passage, and the ZI
cells were treated with 200 µM Zn for 4 h before harvesting. Compared
with results for untreated normal cells, ZI treatment did not affect
the DNA content per plate (data not shown) but significantly increased
the cellular Zn level to 312% of the control level (Table 1).
Consistent with the changes in cellular Zn levels, MT-II mRNA and
apoA-I mRNA abundances were also elevated 24- and 5-fold, respectively,
in ZI cells (Fig. 6).


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Fig. 6.
Effect of short-term Zn induction on cellular MT-II mRNA and apoA-I
mRNA levels in Hep G2 cells. Cells were cultured in normal medium until
end of passage and then were treated with 200 µM Zn for last 4 h.
MT-II and apoA-I mRNA abundances were measured by RNase protection
assay, with 18S rRNA as reference. Values are means ± SE from 4 experiments. Means with different letters are significantly different
(P < 0.05 by 1-way ANOVA). CT,
control.
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DISCUSSION |
Previously, we have demonstrated that moderate Zn deficiency resulted
in marked reductions in plasma total HDL apoA-I levels in rats and
hamsters (18). In addition, the hepatic apoA-I mRNA abundances,
together with the hepatic MT-II mRNA levels, were also downregulated by
Zn deficiency in both rats and hamsters. Furthermore, a 2-day
replenishment with a Zn-adequate diet of Zn-deficient animals
normalized apoA-I levels in plasma and hepatic mRNA abundance to values
that were the same as those for control hamsters and higher than those
for control rats. These observations strongly suggested
that Zn status regulated the hepatic apoA-I gene expression.
In this study, we have developed three methods to deplete cellular Zn
from Hep G2 cells. The Opti-MEM system could deplete cellular Zn from
Hep G2 cells, as well as from BHK cells (13), within one passage.
However, when the treated Hep G2 cells were passed into the next
passage, growth retardation was apparent after 3 days of culture in the
second passage, regardless of the Zn concentration in the media (data
not shown). These observations suggested that the Opti-MEM medium
lacking Zn should only be used for certain applications and may not be
suitable for a transfection assay in the second passage of future Zn
depletion studies. This problem was largely avoided in the study using
Chelex 100, in which 10% extracted serum was used. When cells were
cultured in ZA4 medium, no morphological difference was observed over
three continuous passages, compared with the cells cultured in normal medium (data not shown). In certain situations, a rapid short-term Zn
depletion is desired, and the OP system can fulfill this need.
Besides the chelation of the Zn ion, Chelex 100 is capable of
sequestering other divalent metals. However, the majority of these ions
are toxic heavy metals, which should not exist in FBS or should exist
at very low levels. To reduce the sequestering of other essential
metals, only serum, not the final medium, was extracted. Moreover, all
treatment media used in the Chelex 100 experiment were based on the
same basal medium containing 10% extracted serum, and the only
difference among all treatment media was the level of Zn added.
Although the removal of other metals caused by Chelex 100 extraction
may result in some possible influence on cellular metabolism, this
influence should exist for all cells regardless of the treatment
groups. Thus the observed treatment difference should be solely
contributed by the Zn status.
Whenever cells are directly exposed to a chelator, possible side
effects are major concerns. To address the question of whether the
observed alterations in OP-treated cells were due to the Zn depletion
or due to the side effects of OP, a predetermined amount of 40 µM Zn
(Fig. 2) was added to the OP medium (200 µM OP) to counteract the Zn
depletion effect. The cellular Zn and DNA contents, as well as the
MT-II and apoA-I mRNA abundances, in OP-Zn-treated cells were all
comparable to those for control cells, which were not exposed to
OP. Thus the decreased cellular apoA-I and MT-II mRNA
abundances associated with OP-treated cells were not caused by side
effects of OP other than metal chelation. Similar to Chelex 100, OP has
been reported to be capable of chelating other metals, although it is
widely used as a Zn chelator. It is possible that the chelation of
other ions may also contribute to the observed reduction of the apoA-I
mRNA level in OP cells, and the addition of Zn to OP medium may block
the chelation of both Zn and other ions by OP. To test this
possibility, cellular Cu levels were also determined in the OP
experiment, because cellular Cu status is known to affect the apoA-I
mRNA abundance. No difference in cellular Cu status among control, OP-,
and OP-Zn-treated groups was detected (data not shown). In addition, we
have also used as low as 20 µM Zn in another OP-Zn treatment during a
preliminary study, and this dose was already capable of partially
normalizing the reductions in cellular Zn, MT-II, and apoA-I mRNA
abundances caused by OP (data not shown). Therefore, the observed
reduction of the apoA-I mRNA level in OP cells may be considered to
have resulted mainly from Zn depletion.
Although the three methods used were largely different, Zn depletion by
these methods resulted in marked reductions in cellular Zn, MT-II mRNA
(not measured in the Opti-MEM experiment), and apoA-I mRNA levels.
Moreover, Zn repletion was able to normalize all changes observed in
Zn-depleted cells. These findings were consistent with the in vivo
observations in Zn-deficient and Zn-replete rats and hamsters (18).
Zn status may regulate the apoA-I gene expression at several possible
steps. In our previous studies, depletion of cellular Cu was found to
increase the cellular apoA-I mRNA abundance in Hep G2 cells (19). This
regulation was found to occur mainly at the transcription step, because
the transcription rate measured by nucleus run-off assay and the
promoter activity measured by reporter gene transfection were markedly
increased by the Cu depletion. In the present study, 200 µM Zn
treatment was able to elevate the cellular apoA-I mRNA abundance to
fivefold that of controls within 4 h. The half-time for apoA-I mRNA in
normal Hep G2 cells was estimated to be ~44.5 h (19), and any
possible changes in apoA-I mRNA stability should not have resulted in a
large accumulation of mRNA within a short period of 4 h. Therefore, the
elevated apoA-I mRNA abundance observed in the high-Zn-induced Hep G2
cells must have resulted from a large increase in transcription rate.
The metal-induced MT expression is mediated by the presence of multiple
copies of metal responsive elements (MREs) in their promoters (6).
These MRE sequences are small imperfect motifs, with a consensus MRE
sequence of C
GGCC (core sequence is
underlined) in either orientation. Several nuclear factors are able to
bind to MREs, and such bindings are enhanced by Zn (2). Besides the MT
genes, a number of potential candidate genes responsive to metal
regulation have been reviewed recently (4, 16). The initial search of
the rat apoA-I promoter revealed a DNA sequence with a high degree of
homology to the MRE consensus sequence (4). In addition, our laboratory
has analyzed the human apoA-I promoter by using Transcription Element
Search Software (TESS) against the database of known transcription
factors. Several locations in the human apoA-I promoter were found to
be capable of being recognized by known transcription factors, which
mediated metal regulation. The DNA sequences around the positive
transcription factor binding sites were further compared with the
consensus sequence of MRE. One perfect MRE site and seven MRE-like
sequences (with one mismatch) were identified (data not shown) as a
cluster within the proximal apoA-I promoter (up to ~330
bp), similar to the MREs found in MT genes. The effect of
cellular Zn status on interactions between these MRE-like regions and
nuclear proteins needs to be analyzed by a gel mobility shift assay. In
addition, the influence of cellular Zn status on the activity of the
apoA-I promoter, with or without mutations at these MRE-like sites,
also needs to be examined by a transfection assay.
In this study, both short-term Zn induction and long-term Zn
supplementation altered the cellular apoA-I mRNA abundances, which were
similar in trends but different in magnitudes. These observations
suggested that a similar mechanism(s) may be involved in both cases.
Thus short-term Zn induction can be used as an extension of a Zn
repletion or supplementation system to provide a larger treatment
effect, which may aid in the elucidation of the mechanism(s)
responsible for the regulation of apoA-I gene expression by Zn status.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. Y. Lei,
Dept. of Nutritional Sciences, 309 Shantz Bldg., Univ. of Arizona,
Tucson, AZ 85721 (E-mail: kai{at}u.arizona.edu).
Received 7 December 1998; accepted in final form 17 May 1999.
 |
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