1 Department of Nutritional Sciences, University of Arizona, Tucson, Arizona 85721; and 2 Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742
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ABSTRACT |
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The influence of zinc status on the levels of p53, as well as downstream targets of p53 in cell repair and survival, was examined in human aortic endothelial cells (HAECs). A serum-reduced low-zinc medium (ZD) was used to deplete zinc over one passage. Other treatments included zinc-normal control (ZN), zinc-adequate (ZA), and zinc-supplemented (ZS) treatment with 3.0, 16.0, and 32.0 µM zinc, respectively. Cellular zinc levels in the ZD cells were 64% of ZN controls; levels in the ZA cells were not different, but levels in ZS cells were significantly higher (40%) than in ZN cells. No difference in p53 mRNA abundance was detected among all treatments; however, p53 nuclear protein levels were >100% higher in the ZD and ZS cells and almost 200% higher in the ZA cells than in ZN controls. In addition, p21 mRNA abundance, a downstream target of p53 protein, was increased in the ZS cells compared with both the ZN control and ZD cells. In the ZS cells, bax and mcl-1 were also ~50% higher compared with ZN controls, whereas bcl-2 mRNA was increased compared with ZA cells. Moreover, caspase-3 activity of ZD cells was not different from that of ZN controls but was reduced to 83 and 69% of ZN controls in ZA and ZS cells, respectively. Thus p53 protein and p53 downstream target genes appeared to be modulated by intracellular zinc status in HAECs.
p53 tumor suppressor gene; apoptosis; zinc depleted; zinc supplemented; atherosclerosis
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INTRODUCTION |
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MAINTENANCE OF THE ENDOTHELIUM is important in the prevention of atherosclerosis. Without this maintenance, endothelial dysfunction and injury as well as barrier disruption can initiate and promote atherosclerosis (2, 3). Studying cultured human aortic endothelial cells (HAECs) is useful in understanding mechanisms that involve endothelial cells in atherosclerosis and wound healing (3), allowing for the study of physiological treatments under controlled conditions (9).
Zinc has been shown to be vital to endothelial cells because of its role in the stabilization of membranes (1), endothelial barrier defense (15), maintenance of cellular integrity (13), and prevention of oxidative stress-induced damage (13). In zinc-deficient endothelial cells, barrier function is significantly decreased, and with zinc resupplementation, barrier integrity is restored (15). Zinc inhibits tumor necrosis factor (TNF)-induced damage in endothelial cells (16) as well as inhibits the detrimental effects of oxidized low-density lipoprotein particles (40), a prominent factor in endothelial injury and plaque formation.
The human tumor suppressor gene, p53, plays a role in atherosclerosis (17, 18, 35). p53 functions as a transcription factor that regulates DNA repair, cell proliferation, and cell death. It has been shown to upregulate the bcl-2 family member bax (27), an apoptotic inducer, and to downregulate bcl-2, a cell survival signal. p53 also regulates p21, a cyclin-dependent kinase inhibitor that is involved in cell growth arrest (5, 39). In addition, the wild-type p53 protein has been shown to accumulate in human atherosclerotic tissue (17, 35), and this increased p53 protein may mediate cell proliferation or apoptosis in atherosclerosis (11, 20). With increased p53 accumulation in atherosclerotic tissue, the cyclin-dependent kinase inhibitor p21 is upregulated as well (19). Researchers have also reported an accumulation of p53 in human atherosclerotic plaques with increased apoptosis as well (18, 21). During endothelial cell apoptosis, mitochondrial cytochrome c is released into the cytosol. Moreover, bax levels in the cytosol are decreased, and caspases-2, -3, -6, -7, -8, and -9 are activated (10). However, the role of apoptosis and p53-dependent apoptosis in atherosclerosis is still unclear.
Zinc has been shown to play a structural role in p53 (12) and is imperative for its stability as well as its DNA binding activity. When wild-type p53 is exposed to zinc chelators or divalent cation resins, the wild-type protein adopts a mutant conformation and the ability of p53 to bind DNA decreases (12, 31). In zinc-depleted Hep G2 cells, p53 mRNA abundance and p53 nuclear protein levels were increased, indicating that zinc has an influence in p53 gene expression (32). The role of zinc in p53 expression is an important one (6) and may have an impact on endothelial cell integrity and its response to injury or stress, such as with zinc deficiency.
Impaired endothelial cell metabolism may compromise barrier function and endothelial cell integrity, causing implications in pathogenesis of atherosclerosis by increasing endothelial cell turnover (14). Zinc may regulate apoptosis both in vivo and in vitro as well as modulate apoptotic regulators in the cell death pathway (4, 7). Zinc supplementation has been found to inhibit caspase-3 activity, a major executioner of apoptosis, as well as other caspases involved in apoptosis (29, 36). In addition, zinc has been shown to increase the ratio of bcl-2 to bax in U-937 cells pretreated with hydrogen peroxide. This increased ratio is an indicator of enhanced cell survival (8). Ultimately, zinc can act as a regulator of mitosis and apoptosis in monitoring tissue growth (4).
The prevalence of subclinical zinc deficiency has been reported in certain segments of the U.S. population (34). These alterations in dietary zinc status may have ramifications on p53, regulators of apoptosis, and, ultimately, the development of atherosclerosis. The objective of the present work was to determine the influence of cellular zinc status on p53 protein and p53 downstream target genes involved in DNA repair, cell cycle arrest, and apoptosis in HAECs and its possible implications on atherosclerotic development.
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MATERIALS AND METHODS |
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Cell culture and treatment media. The normal human aortic endothelial cell line, HAECs, was purchased from Clonetics (San Diego, CA) and was used as a representative of human aortic cell behavior in response to different levels of zinc. All reagents used in cell culture were obtained from Clonetics and Life Technologies (Grand Island, NY). HAECs were maintained in Clonetics' recommended endothelial growth medium (EGM), which included 10 ml of fetal bovine serum (FBS), 3 g/l bovine brain extract, 1.0 g/l hydrocortisone, 10 mg/l human recombinant epidermal growth factor, 50 g/l gentamicin, and 50 mg/l amphotericin B. Media were replaced every other day. Cells reached confluency in ~10 to 10.5 days, and nearly confluent cells were subcultured with trypsin-EDTA at a ratio of 1:8. The HAECs were subcultured for two passages to increase cell numbers and were then cultured in experimental media.
A zinc-free EGM basal medium, in which Clonetics omitted the addition of ZnSO4, was used as the zinc-depleted (ZD) medium. This zinc-free medium consisted of endothelial basal medium with the added growth components and contained residual amounts of zinc (0.8 µM) as detected by flame atomic absorption spectrophotometry. The zinc-free basal medium of 0.8 µM ZnSO4 was suitable without affecting overall growth in HAECs and was used as the ZD medium. For the other three treatment groups, zinc was added to the media in the form of ZnSO4 so that the only difference between these media was the zinc concentration. For zinc-normal (ZN) medium, 3.0 µM ZnSO4 was added to the zinc-free basal medium; the zinc-adequate (ZA) medium contained 16.0 µM ZnSO4; and the zinc-supplemented (ZS) medium contained 32.0 µM ZnSO4. The ZN medium was used as the control for experiments because the zinc concentration is comparable to that of standard culture medium. The ZA group was used as a representative of human plasma zinc levels, and the ZS group was used for comparison to human plasma zinc levels attainable by oral supplementation (37). After HAECs were subcultured, the cells were cultured overnight in ZN medium, assigned to one of the four treatment groups, and changed to their respective medium. Cells were then grown in ZD, ZN, ZA, or ZS medium for 10 days (one passage) and harvested for cellular zinc and DNA content, total cellular RNA isolation, nuclear protein extraction, and cell lysate isolation.Cellular zinc and DNA content determination. Both cells and media were collected by scraping from 100-mm tissue culture plates. Cell suspensions were then centrifuged at 500 g for 5 min at 4°C, and cell pellets were washed twice with phosphate-buffered saline (PBS). Cells were resuspended into 1.5 ml PBS and sonicated for two 30-s intervals. An aliquot of the sonicated cell suspension was used to measure cellular zinc content by flame atomic absorption spectrophotometry (Hitachi, San Jose, CA). Zinc standard solutions (Fisher Scientific, Fair Lawn, NJ) ranging from 0.05 to 1.0 ppm were used to generate a linear standard curve. The zinc content of the cells was determined on the basis of these zinc reference solutions. In addition, the certified zinc solutions were compared with bovine Liver Standard Reference (U.S. Department of Commerce, National Institute of Standards, Gaithersburg, MD). Appropriate blanks were employed for all measurements. From the same sample, a small aliquot of the sonicated cell suspension was used to measure cellular DNA content (41, 42). Cellular zinc per microgram of DNA is shown, because a linear relationship between cellular DNA and cell number was previously established (42).
Western blot analysis. Nuclear extracts were prepared as previously described by Reaves et al. (32). Nuclear extract (5 µg) was combined with an equal volume of sample loading buffer (20% glycerol, 10% 2-mercaptoethanol, 5% SDS, 200 mM Tris · HCl, pH 6.7, and 0.01% bromphenol blue), boiled for 3 min, and then subjected to 10% SDS-PAGE (Bio-Rad, Hercules, CA). After electrophoresis, gels were briefly equilibrated in transfer buffer (20% methanol, 192 mM glycine, 25 mM Tris-aminomethane, and 0.05% SDS) before being transferred onto nitrocellulose membranes. Transfer was performed at 30 V overnight at 4°C. Equal loading of samples was verified by staining a duplicate gel with Coomassie brilliant blue R-250 and scanning with a laser densitometer to compare optical density units between lanes. After membranes were blocked [10 mM Tris · HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20 (TBST) with 5% nonfat dry milk] for at least 1 h, blots were then incubated with mouse anti-p53 antibody DO-1 (Santa Cruz Biotech, Santa Cruz, CA) diluted to 0.2 µg/ml in TBST with 5% nonfat dry milk at 4°C overnight, followed by four 10-min washes in TBST. Incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Santa Cruz Biotechnology), diluted to 0.25 mg/l in TBST with 5% nonfat dry milk, was performed for 1 h, followed by four 10-min washes in TBST. Autoradiography was performed by utilizing enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Amersham, Arlington Heights, IL). p53 bands were verified by running human p53-glutathione S-transferase (GST) fusion protein (Oncogene Research Products, Cambridge, MA) on a lane in each gel. Blots were also stained with amido black and photographed to document equivalent protein loading. Laser densitometry (Molecular Dynamics, Sunnyvale, CA) was used to quantify p53 bands after linearity curves were established.
RNase protection assay.
Total cellular RNA was isolated from HAECs by using the RNAqueous kit
(Ambion, Austin, TX) according to manufacturer's instructions, and the
integrity of the RNA was verified by electrophoresis. The mRNA
abundance of human apoptotic genes including p53, gadd45, p21,
c-fos, bax, bcl-x, bcl-2, and mcl-1 were measured by RNase protection assay (RPA) using the human-stress1 multiprobe templates (Pharmingen, San Diego, CA). The human L32 and
glyceraldehyde-6-phosphate dehydrogenase probes were also included in
the multiprobe and were used as internal references for normalization.
Labeled riboprobes were synthesized by using the In Vitro Transcription
System kit with T7 RNA polymerase (Pharmingen) and
[-32P]UTP (3,000 Ci/mmol; NEN, Boston, MA).
Caspase-3 activity assay in zinc-treated HAECs. Caspase-3 activity was measured with the ApoAlert CPP32 colorimetric assay kit (Clontech Laboratories, Palo Alto, CA). This method uses a colorimetric assay to monitor cleavage of an Ac-DEVD-p-nitroanilide substrate, which resembles the caspase-3 cleavage site. Cells were cultured in their respective zinc treatments for one passage. Briefly, 2 × 106 cells were lysed in lysis buffer on ice for 10 min. The cell lysates were then centrifuged at 200 g for 3 min at 4°C to precipitate cellular debris. After centrifugation, 50 µl of 2× reaction buffer (Clontech), containing 10 mM dithiothreitol and 50 µM conjugated DEVD-p-nitroanilide substrate were added to the supernatant fractions. The supernatant fractions were incubated at 37°C for 2 h in a water bath and measured in a colorimetric spectrometer at 405 nm. A negative control using 0.5 µl DEVD-fmk, a caspase-3 irreversible inhibitor, was used to show specificity. Samples from Jurkat cells induced with 0.25 mg/l anti-human Fas (clone CH11; Upstate Biotech, Lake Placid, NY) for 16 h were used as a positive control for caspase-3 activity. A parallel control reaction that did not contain conjugated substrate was also used, as was a sample from noninduced Jurkat cells. A calibration curve using concentrations ranging from 0 to 20 nmol of p-nitroanilide was established. Values are expressed as the concentration of Ac-DEVD-p-nitroanilide cleaved over the course of a 2-h incubation interval.
Statistical analysis. Statistical analyses were performed by using the SAS software release General Linear Models (version 6; SAS Institute, Cary, NC). The data were analyzed using one-way ANOVA, and the means were further analyzed by least significant difference. Values are expressed as means ± SE with a statistical probability of P < 0.05 being considered significant.
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RESULTS |
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HAECs responded to zinc treatments with alterations in cellular
zinc levels.
HAECs were cultured for one passage (10 days) in the media containing
different levels of zinc. We purchased basal endothelial medium to
which zinc was not added (Clonetics). The addition of 2% FBS and other
growth factors, which contained residual amounts of zinc as determined
by atomic absorption spectrophotometry (data not shown), increased the
basal level zinc concentration of the medium to 0.8 µM. When the
endothelial cells were cultured directly in this basal medium
containing 0.8 µM zinc, they were able to reach a confluent state
comparable to cells cultured in regular medium of 3.0 µM zinc. This
indicates that cell growth was not affected by the low zinc status of
the basal medium, although cellular zinc levels were depleted. The
higher supplemental levels of zinc used were determined by using a
dosage curve ranging from low to high concentrations of zinc, and DNA
content per plate was utilized as an index of cell growth. From the
zinc-depleted level of zinc to the higher dosage of zinc
supplementation, the level of DNA content was not significantly
different among treatments (Fig.
1A). In contrast, the cellular
zinc content increased in a dose-dependent manner as the level of zinc
in the media increased (Fig. 1A). Furthermore, cells were
cultured for two passages in the four treatment media, and there were
no significant differences in cell growth among groups.
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Nuclear p53 protein was significantly lower in ZN control cells.
Western blot analysis of nuclear extracts was used to quantitate
nuclear p53 protein levels in each treatment. Nuclear p53 protein
levels in ZD cells were significantly higher compared with ZN control
(216.5 ± 40.0 vs. 100 ± 9.3%) (Fig.
2). In addition, the nuclear p53 protein
levels in the ZA and ZS cells were nearly 200 and 100% higher,
respectively, than in the ZN control cells (Fig. 2). Moreover, there
was no significant difference in p53 protein levels among the ZD, ZA,
and ZS cells. p53 mRNA levels were relatively low compared with the
other genes measured, and there was no significant difference among
treatment groups.
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Zinc treatment alters p53 protein downstream genes, p21, bax,
mcl-1, and bcl-2 mRNA abundance.
One of the major downstream targets of the p53 protein, p21, was also
affected by zinc treatment. Although p21 mRNA abundance in the ZD and
ZN cells was not significantly different, it was lower than in the ZS
cells (166.3 ± 14.7%), which were not significantly different
from the ZA cells (122.1 ± 17.2%) (Fig.
3). Bax, an apoptotic inducer and a
downstream target of p53, was not different in the ZD, ZN, or ZA cells;
however, the ZS cells had an ~50% higher mRNA abundance than ZN
control or ZD cells (Fig. 4).
Interestingly, mcl-1 mRNA abundance was also significantly higher in
the ZS group compared with the ZN control and ZD cells (140.6 ± 17.0, 100.0 ± 14.5, and 73.4 ± 5.9%, respectively) but was
not different from the ZA cells (Fig. 5).
Bcl-2 mRNA abundance was increased in the ZS cells compared with the ZA
cells (138.9 ± 6.9 vs. 94.2 ± 19.9%) but was not
significantly different from the ZD and ZN control cells (Fig.
6). The abundances of bcl-x,
c-fos, and gadd45 were not significantly different among
treatments.
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Caspase-3 activity was depressed with higher zinc concentrations.
Caspase-3 activity was measured in endothelial cells exposed for one
passage in different zinc treatments. In the ZD cells, caspase-3
activity was not significantly different from that in the ZN control
cells (Fig. 7). However, caspase-3
activity was reduced in the ZA and ZS cells to 83 and 69% of that in
control ZN cells, respectively (Fig. 7). Interestingly, with increasing concentrations of zinc, caspase-3 activity was decreased, which has
been shown to occur in other studies using different cell model systems
(8, 24).
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DISCUSSION |
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Because endothelial cells are constantly exposed to not only damage-inducing agents but also protective nutrients, cellular zinc may serve to protect endothelial cell integrity and, ultimately, atherosclerosis. HAECs have been used as a model to study the influence of factors, both protective and damaging, on atherosclerotic development. Because of our ability to deplete cellular zinc from HAECs, we believe this model provides important information as to how zinc may affect p53 protein as well as p53 target genes and their possible involvement in endothelial cell function.
In these studies, our depletion of cellular zinc did not involve the use of any cell permeable chelators or a chelating resin. Instead, the zinc was excluded during preparation of the basal medium, which provided an effective, yet noninvasive technique to control the levels of zinc in the media. With the use of this low-zinc medium, 2% FBS (as recommended by manufacturer) was added back as well as other growth factors that increased the zinc concentration to 0.8 µM (compared with 3.0 µM for normal medium). Cells cultured in the medium depleted of zinc for two consecutive passages did not appear to be morphologically distinct from control cells cultured in normal medium. The growth rate and viability of the zinc-depleted cells were not altered in comparison control groups. We believe this depletion of zinc may be relevant in view of marginal zinc-deficient states seen in certain subpopulations of the U.S. (33).
HAECs are moderately responsive to alterations in zinc concentrations in the media. The ZD cells had significantly lower zinc levels compared with ZN and ZS cells. The ZN control cells were not significantly different from the ZA cells, possibly because of the adaptation of cultured cells to the levels of zinc found in media in vitro, as well as their conditioned response to sufficiently handle the levels found in human plasma in vivo. With increasing zinc levels, DNA content remained relatively the same, indicating that differences in cell growth between treatment groups did not occur.
In previous studies, we have found that p53 mRNA is induced in zinc-depleted Hep G2 cells (32). However, in the present study, the p53 mRNA abundances were too low in HAECs to be quantitated accurately among treatments. Perhaps no differences in mRNA abundance could be found in the present study because zinc depletion was less drastic or because the p53 mRNA response may be cell type specific. Most studies have indicated that p53 is mainly regulated at the protein level, such as posttranslational modification, localization, and degradation (28). Normally, wild-type p53 protein and mRNA levels are low and found in small quantities; however, when induced, the response is posttranscriptional (22). Once signaled, levels of p53 increase, and p53 is converted from its inactive to its active form and then translocates and accumulates in the nucleus. The process of nuclear accumulation is a cell type-specific process (25), and mRNA levels are highly variable from one cell type to another.
Our data demonstrate that nuclear p53 protein levels were higher in the ZD, ZA, and ZS cells compared with ZN control cells. With zinc depletion, cells may be undergoing oxidative stress (14), and this could result in changes in protein-zinc or protein-DNA interactions that involve cysteines (38). Oxidative stress can result in p53 adopting a mutant conformation that is unable to bind to DNA (12). Marginal zinc deficiency could have induced stress, whether oxidative or cell proliferative, in the HAECs and provoked signals that resulted in a posttranscriptional upregulation of p53. Although there was no difference in cell growth and viability in the ZS group, the increased p53 protein induced relatively small increases in p53 downstream targets, including p21, bax, and bcl-2 mRNA, and also induced mcl-1 mRNA. In ZD cells, the elevated p53 protein may have resulted from an impaired degradation process facilitated by an increase in the c-Jun NH2-terminal kinase (JNK) phosphorylation of p53, leading to an inhibition of Mdm2 association and p53 ubiquitination as well as prolonged p53 half-life (7a). This contention is supported by our recent findings in Hep G2 cells of an increased JNK activity in ZD compared with ZN cells (unpublished data). Other studies have shown that when zinc chelators are used to induce severe zinc deficiency in cells, p53 protein confirmation is altered, thus impairing the ability of p53 to bind to its responsive elements in downstream target genes (12, 31). Reductions in cellular zinc levels may also have induced a mutated p53 conformational change in the ZD HAECs that impaired the ability of p53 to bind DNA and to act as a transcriptional factor to enhance promoter activity of p53 target genes. This contention may explain why our mRNA data for certain p53-regulated genes are not responsive to the observed elevation of p53 level. For example, with zinc depletion, p53 protein was increased; however, p21 mRNA remained unaffected. Nevertheless, this may explain why cell growth was not affected in these cells, because p21 is a proapoptotic factor. Currently, studies are being conducted in our laboratory to examine the influence of zinc status on the confirmation and turnover of p53 protein in these cells.
In ZA and ZS cells, the increase in p53 may have resulted from an enhanced p53 stabilization leading to a general reduction in degradation. Current studies being conducted in our laboratory to examine the influence of zinc supplementation on p53 tetramerization in the nucleus will indicate whether p53 stabilization is the responsible mechanism. In ZS cells, the twofold increased p53 protein level induced relatively small increases in p53 downstream targets, including p21, bax, and bcl-2 mRNA and also mcl-1 mRNA. These changes were associated with no changes in cell growth and viability. Although the cells appeared viable and morphologically normal, these changes in gene expression may be indicative of increased cell turnover for maintenance of normal cell growth and stabilization of archetypal cell population. Interestingly, the increase in nuclear p53 protein did not affect cell growth or apoptosis, two of the major regulatory processes influenced by p53. However, p53 has also been shown to be involved in other cellular functions of the cells such as targeting regulatory genes involved in oxidative stress response as well as other cytotoxic stresses (22). Because endothelial cells are constantly exposed to adverse conditions such as free radicals, changes in cellular zinc status may play a vital role in triggering these stresses. Moreover, p53 has been shown to be sensitive to oxidative stress because of specific cysteine residues that are within the zinc-finger-like motif and DNA binding domain (12). These changes in p53 protein may be significant in these endothelial cells. However, further studies need to be performed to examine the protein levels of p53 target genes as well as overall functionality of p53. Additional studies are also needed to examine whether zinc may alter HAEC cell death, dysfunction, cell cycle, or proliferation beyond one or two passages. In this study, we have shown that zinc, whether depleted or supplemented, alters normal p53 expression and that this change in cellular zinc status, as seen in certain segments of the population, may enhance or reduce p53 and its ability to protect cells from physiological stresses.
p21 mRNA was increased in the ZS group. Normally, induction of p21 mRNA inhibits cyclin-dependent kinases and allows for cell cycle arrest at the G1 phase. Once cells arrest at the G1 phase, damage to DNA can be repaired. With a high level of zinc in the HAECs, and the increased p53 protein, p21 mRNA may be transcriptionally upregulated in response to injury; however, we did not observe growth arrest. Other researchers have shown that p21 mRNA was upregulated in human prostate carcinoma cell lines when treated with zinc (23). These results are surprising; however, these cells may be more sensitive to changes in cellular zinc compared with an in vivo system, which can be difficult to mimic in a more artificial system such as cell culture. Perhaps the level of zinc used in the supplemented groups is potentially stressful to endothelial cells, which are more sensitive to zinc changes. This stress may induce p53 protein levels, which can upregulate downstream target genes, such as p21. With zinc supplementation, DNA synthesis is increased in human cell lines (30). Thus the normal cell growth observed in the supplemented HAECs may have resulted from a balance between enhanced synthesis and repair of DNA. Further studies need to examine electrophoretic mobility shift assays and transfection experiments performed under zinc supplementation conditions to identify how zinc affects p53 binding to responsive elements within p21 promoter sequences (5). Furthermore, we have previously seen that within the human partial promoter region of p21, there are potential metal-responsive elements (MREs) (unpublished results). These elements have been shown to be regulated by zinc (4), and this regulation may play a role in the transcription of p21. Whether these MREs are functional and responsive to zinc is unknown; however, we found that with higher cellular zinc levels, p21 mRNA expression was increased.
Bax mRNA was also increased in the ZS group. This bax gene is a proapoptotic member of the bcl-2 family and is a p53 target gene whose expression can be upregulated through a p53 DNA-binding response element in its promoter (27). Previously, it has been shown that zinc supplementation inhibits apoptosis (4) as well as bax protein (8). Our results indicate that bax mRNA was increased; however, we did not evaluate bax protein levels or translocation of bax to the mitochondria. Furthermore, endothelial cell apoptosis is associated with decreased levels of bax in the cytosol (10). mRNA levels may not be an accurate estimation of bax protein levels in the cytosol or mitochondria. Bax mRNA abundance may also be enhanced to promote dimerization with bcl-2, and this dimerization is required for bcl-2 to block apoptosis (43).
Zinc supplementation increased both mcl-1 and bcl-2 mRNA, both of which are cell survival genes. However, p53 protein has been shown to repress bcl-2 (26). These findings are similar to that reported by Fukamachi et al. (8), who found that zinc supplementation increased bcl-2 protein and the bcl-2-to-bax ratio. Bcl-2 and bax Western blot analyses are needed to determine whether the level of zinc used in our study affects the protein levels, as well as the ratio of bcl-2 to bax.
Interestingly, our levels of zinc for both ZA and ZS did cause caspase-3 activity to be reduced compared with controls. This follows the findings of other studies that zinc supplementation decreased caspase-3 activity and its cleavage as well as overall apoptosis (8, 24). Although the ZS group had increased bax mRNA, all other indicators suggest cell survival, including decreased caspase-3 activity and increased bcl-2, mcl-1, and p21 mRNA.
Our findings demonstrate that p53 protein and downstream targets and events of increased p53 were responsive to depleted or elevated cellular zinc status. We proposed that in zinc-depleted HAECs, a portion of this elevated p53 protein may assume mutant confirmation, which becomes less effective in modulating downstream target genes. Similarly, we have observed increases in nuclear p53 protein levels in zinc-deficient normal bronchial epithelial cells(6) and in Hep G2 cells (32), which were accompanied by no reduction in cell growth. However, in severely zinc-deficient Hep G2 cells, a reduction in cell growth was accompanied by a depressed progression from G1 to S phase (5). Thus a severe zinc-deficient state may enhance endothelial dysfunction, and with depressed ability of p53 to promote genome stability, endothelial barrier disruption may enhance atherosclerosis. In addition, our results also imply that with higher zinc supplementation, p53 protein levels accumulate and induce p53 target genes involved in DNA repair and cell survival. These changes were accompanied by the repressed activity of caspase-3, which may promote repair mechanisms in endothelial cell homeostasis. At present, much work remains to be performed to elucidate the mechanism responsible for the modulation of p53 protein and of regulators of cell turnover and repair in the development of atherosclerosis by cellular zinc status.
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ACKNOWLEDGEMENTS |
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This work was supported by the Arizona Disease Control Research Commission (S. K. Reaves), US Department of Agriculture National Research Initiative Competitive Grant 96-35200-3248, and funds from the University of Arizona Agricultural Experiment Station (K. Y. Lei).
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Y. Lei, Dept. of Nutrition and Food Science, Univ. of Maryland, 3304 Marie Mount Hall, College Park, MD 20742.
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. Section 1734 solely to indicate this fact.
April 18, 2002;10.1152/ajpcell.00248.2001
Received 4 June 2001; accepted in final form 16 April 2002.
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