From the Department of Molecular and Cellular Biochemistry, The Ohio State University, College of Medicine, The Ohio State University, Columbus, Ohio 43210
Received for publication, March 20, 2003 , and in revised form, April 23, 2003.
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ABSTRACT |
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INTRODUCTION |
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The molecular mechanism(s) for the Cr6+-mediated toxicity has
not been fully elucidated. The chromium intermediates appear to interact
directly with cellular constituents that lead to generation of reactive oxygen
species (8,
9). Chromium treatment results
in DNA strand breakage (10,
11) and DNA-protein
cross-linking in vivo
(12,
13). Reactive oxygen species
generated during intracellular reduction of Cr6+ affects almost
every aspect of cellular function. Damage to cellular macromolecules and
aberrations in gene expression ultimately lead to apoptosis or necrosis in the
majority of the cells and uncontrolled cell proliferation in a few, causing
cancer. Apoptosis induced by Cr6+ treatment in lung epithelial
cells involves both p53-dependent and p53-independent pathways
(14,
15). Cr6+ also
activates a stress response protein (NF-B), which in turn activates
anti-apoptotic proteins (16),
thus protecting cells from apoptotic cell death.
Our laboratory has been studying the molecular mechanisms of heavy
metal-induced expression of metallothionein genes
(17). Metallothionein I and II
(MT-I1 and MT-II)
belong to a family of low molecular weight, cysteine-rich, and high
metal-containing (as metal-thiolate clusters) stress response proteins that
protect cells not only from heavy metals but also from reactive oxygen species
(18,
19). Metallothioneins induced
in response to heavy metals like zinc, cadmium, copper, mercury, gold, silver,
cobalt, nickel, and bismuth act as scavengers of these toxic metals and help
maintain zinc and copper homeostasis
(17,
20). It was of interest to
examine the induction of these proteins by chromium as a means to scavenge the
toxic form of chromium. Two other members of this family of proteins, MT-III
and MT-IV, are expressed in a tissue-specific manner in brain and in squamous
epithelial cells of tongue and skin, respectively. Several metal regulatory
elements (MREs) located on the immediate MT promoter mediate its
robust expression by recruiting the key transcription factor metal-responsive
transcription factor 1 (MTF1). MTF1 is a 7184-kDa protein with six zinc
fingers of the Cys-2His-2 type and three different transactivation
domains, all of which function cooperatively
(21). In response to heavy
metals MTF1 translocates to the nucleus, attains a conformation that can bind
to the cognate cis elements, and transactivates the gene. Among the different
heavy metals only zinc can directly activate MTF1, whereas other metals like
cadmium probably activate it by mobilizing the intracellular zinc pool
(22). MTF1 is essential for
development as knockout mice die because of liver decay
(23). Recent studies show that
MTF1 has several important target genes, such as those for
C/EBP (24),
the placental growth factor (PlGF)
(25), and zinc transporter-1
(ZnT-1) (23). The
molecular mechanism by which MTF1 is activated in response to different
stimuli, triggering its DNA binding and subsequent transactivation of the
target genes, remains to be elucidated. Recent studies show that various
signal transduction pathways that include protein kinase C, casein kinase II,
and tyrosine kinase modulate the transactivation function but not the DNA
binding activity of MTF1 (26).
Besides MTF1, several other transcription factors like Sp1, USF1,
glucocorticoid receptor, STAT3, are also involved in MT gene expression in
response to different physiological and pathological conditions
(27,
28). In the present study we
have identified potential toxin hexavalent chromium, the predominant form in
the industrial waste, that not only fails to induce MT expression but also
interferes with its expression in response to other heavy metals by inhibiting
the transactivation function of MTF1.
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MATERIALS AND METHODS |
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Construction of Retroviral Vector Harboring Human MTF1 and Generation of Stable Cell Lines Expressing MTF1To construct recombinant human MTF1 cDNA with the FLAG tag at the C terminus, we amplified the MTF1 coding region from pchMTF1 with the primers F, 5'-TAATACGACTCACTATAGGGAGACCC-3', and R, 5'-ATCTTATCATTATCATTTGTCATCGTCGTCCTTGTAGTCCTTGGAGAAGCTGCTGGTGAG-3', and ligated the PCR product to the SnaB1 site of the pBabe-puro vector (29). pBabe-hMTF1-FLAG was then transfected into dko-7 (double knock out for hMTF1), cells and the cells overexpressing hMTF1-FLAG were selected with DMEM containing puromycin (4 µg/ml) for 14 days. The individual clones were picked up by cloning rings and allowed to grow in the same medium. The level of hMTF1-FLAG expression in different clones was monitored by electrophoretic mobility shift assay of the complex formed between MRE-s oligo and the whole cell extract prepared from dko-7 and different recombinant clones. MTF1 forms specific complex with MRE-s oligo (30).
Western Blot Analysis with Anti-FLAG AntibodiesThe whole cell extracts (150 µg protein) were separated by SDS-PAGE (10% acrylamide), transferred to nitrocellulose membrane, and subjected to immunoblot analysis with the anti-FLAG M2 antibody (Sigma) following the manufacturer's protocol.
Isolation of RNA and Northern Blot AnalysisTotal RNA was
isolated from different cell lines by the guanidine thiocyanate acid phenol
method (31). Twenty-five or
thirty micrograms (as indicated in the legend of Fig.
1B,
2 and
4) total RNA was
separated by formaldehydeagarose (1.2%) gel electrophoresis and transferred to
a nylon membrane. The membrane was then hybridized with
[-32P]dCTP-labeled mouse MT-I mini gene
(pMT-I
i)
(32). The blots were subjected
to autoradiography as well as PhosphorImager analysis to quantify
32P signal in each lane using the volume analysis program
(Molecular Dynamics). To measure the amount of RNA loaded onto each lane, the
blots were stripped of MT-I probe following the manufacturer's protocol and
then re-probed with random-primed [
-32P]dCTP-labeled rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, a housekeeping gene
(33).
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RT-PCRFor RT-PCR, reverse transcription was carried out
with random hexamers (PerkinElmer Life Sciences) and murine leukemia virus
reverse transcriptase from 1 µg of total RNA following the protocol
provided in the GeneAmp RNA PCR kit (PerkinElmer Life Sciences). One-tenth or
twentieth of the RT reaction was subsequently PCR-amplified for each of the
genes of interest with dNTP (Roche Applied Science) and Taq
polymerase (Invitrogen). PCR amplifications were also performed with reverse
transcriptase minus RT mixes to rule out genomic DNA contamination (not
shown). Gene specific primers used for amplification of the human MT-IIA mouse
ZnT-1 and human -actin cDNA are as follows: hMT-IIA-F,
5'-TCCTGCAAATGCAAAGAGTG-3'; hMT-IIA-R,
5'-TATAGCAAACGGTCACGGTC-3'; mZnT-1-S,
5'-TGACAATCTGGAAGCGGAAGACAAC-3'; mZnT-1-A,
5'-GGAAGCGGGGTCCTCACATTTTATG-3';h
-actin-F,
5'-TTTGAGACCTTCAACACCCCAGCC-3'; h
-actin-R,
5'-AATGTCACGCACGATTTCCCGC-3.
Nuclear Run-on TranscriptionNuclei from 108
cells were isolated from cells by homogenization in sucrose buffer I (0.32
M sucrose, 2 mM Mg(OCH3CO)2, 0.1
mM EDTA, 10 mM Tris-HCl (pH 8.0), 1 mM
dithiothreitol, and 0.5% Nonidet P-40)) and mixed with equal volume of sucrose
buffer II (2 M sucrose, 5 mM
Mg(OCH3CO)2, 0.1 mM EDTA, 10 mM
Tris-HCl (pH 8.0), 1 mM dithiothreitol). The mixture was layered on
top of 4 ml of sucrose buffer II, and nuclei were collected by spinning at
100,000 x g at 4 °C for 45 min. The pellet was resuspended
in in vitro transcription buffer (400 µl) and, if not used
immediately, stored as 200-µl aliquots in liquid N2. For run-on
transcription nuclei (5 x 107) were incubated with NTPs
containing ([-32P]UTP) for 5 min at 30 °C, and RNA was
purified. The labeled RNA was allowed to hybridize to the nylon membrane
containing empty vector as well as plasmid DNA with different cDNA inserts,
and the signal was determined as described
(19).
Electrophoretic Mobility Shift AssayNuclear extracts used
for the DNA binding activities of MTF1 proteins were prepared as described
(33), incubated with
[-32P]ATP-labeled MRE-s duplex probe in the binding buffer
(20 mM Hepes (pH 7.9), 7590 mM KCl, 5
mM MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.2 µg of poly(dI-dC)/µg of protein, and 1012%
glycerol), and resolved on a 6% non-denaturing polyacrylamide gel
(33). The MTF1 antibody used
for the supershift experiment was a generous gift from Dr. Walter Scaffner.
The Sp1 consensus oligonucleotide and anti-STAT3 antibody (sc-842) were from
Santa Cruz Biotechnology Inc, Santa Cruz, CA.
In Vivo Genomic FootprintingIn vivo DNA methylation and extraction of the methylated DNA were performed as described (34). The mouse MT-I and human MT-IIA promoter were amplified by ligation-mediated PCR (LM-PCR) according to the procedure of Mueller and Wold (35), as modified by Ping et al. (36). Briefly, HepG2 and MTF111 cells in DMEM (control or treated with 100 µM zinc sulfate) were exposed to a limited dimethyl sulfate treatment (1 µl/ml, 2 min at room temperature). The genomic DNA was isolated from the cells, purified, and subjected to piperidine cleavage (10%) at 90 °C for 30 min. The purified cleaved DNA (2 µg) was then subjected to LM-PCR to amplify MT-I promoters using primers described by Mueller and Wold (35). The primers used to amplify MT-IIA promoters are the following: HMT2A/5'-1, 5'-ACCTGTCTGCACTTCCAACC-3'; HMT2A/5'-2, 5'-GCTAACGGCTCAGGTTCGAG-3'; HMT2A/5'-3, 5'-ACGGCTCAGGTTCGAGTACAGG-3' (the annealing temperature for this set of primers was 58, 60, and 63 °C, respectively); HMT2A/3'-1, 5'-CATCCCCAGCCTCTTACC-3'; HMT2A/3'-2, 5'-AAGAGGCGGCTAGAGTCGG-3'; HMT2A/3'-3, 5'-TAGAGTCGGGACAGGTTGCACG-3' (the annealing temperature for the 3'-primers are 56, 60, and 64.8 °C, respectively).
Transfection AssayHepG2 cells were grown in DMEM with 10%
fetal bovine serum. For the transfection assay, 5.0 x 105
cells were plated onto 60-mm dishes 24 h beforehand and then transfected using
the calcium phosphate precipitation method
(37). Each transfection
mixture contained a total of 8.8 µg of DNA including the reporter plasmid
pMT-Luc (38), pRL-TK
(Renilla luciferase reporter driven by HSV-tk promoter, Promega) as
internal control ( the amount of the reporter plasmid), and eukaryotic
expression vector harboring the gene of interest described in the respective
figure legends. The cells were allowed to incubate in the presence of the
transfection mixture in complete medium (DMEM plus 10% fetal bovine serum) for
16 h in a 37 °C incubator with 5% CO2 followed by replacement
with fresh medium. Eight hour after removal of the transfection mixture the
cells were split into 12 35-mm dishes, and each triplicate set was either left
untreated or treated with Zn2+ and/or Cr6+ for 3 h
before harvest. After a total of 48 h (and respective treatments) the cells
were harvested in 1x lysis buffer (Promega), and luciferase activity was
measured using the dual luciferase assay kit (Promega) in a luminometer (Lumat
LB 9507; EG&G Berthold, Oak Ridge, TN). The different glutathione
S-transferase-MTF1 fusion proteins used in the transfection studies
were generous gifts from Dr. Schaffner
(21).
TUNEL and Annexin V AssaysFor detection of events of
apoptosis in cells treated with heavy metals, an in situ cell death
detection kit, fluorescein (Roche Applied Science Biochemicals), and ApoAlert
annexin V kit (BD Sciences) were used. dko-7 and MTF112 cells
were plated in 8-well LabTek chamber slides (Nalgene NUNC International) at a
density of 30,000 cells per well in DMEM containing 5% fetal bovine serum
and puromycin, where appropriate. The cells were allowed to attach to the
chamber and grow overnight in an incubator at 37 °C in 5% CO2.
Apoptosis was induced by treating cells with 100 µM
Zn2+, 30 µM Cd2+, and 100 µM
Cr6+ (as indicated in the Figs.
9 and
10). The cells were then fixed
and permeabilized for TUNEL assay. The assay was performed, and the cells were
stained with propidium iodide (PI) according to the manufacturer's protocol.
For annexin V assay, the cells were induced with the metals as described and
stained with annexin V-FITC and PI according to the protocol provided by the
company.
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MTT AssayTo assess the viability of cells during metal treatment, we performed MTT assay using a kit from Roche Applied Science. MTT reduction was carried out using HepG2 cells. The cells were plated at a density of 0.5 x 104 cells/well in a 96-well plate and allowed to grow overnight followed by treatment with metals (100 µM each) for 2 and 4 h. Cells were then washed with medium, and 200 µl of fresh medium was added in each well followed by the addition of 10 µl of MTT reagent. After 4 h of incubation at 37 °C, 200 µl of lysis buffer was added to each well, incubated at 37 °C overnight, and read using a enzyme-linked immunosorbent assay plate reader at 575 nm.
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RESULTS |
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To investigate the role of Cr6+ on MT expression, we treated HepG2 cells with K2Cr2O7 and performed Northern blots of total RNA with 32P-labeled MT-I cDNA as the probe. The results demonstrated that unlike Cd2+ or Zn2+, Cr6+ by itself could not activate MT gene expression in the cells (Fig. 1B, lanes 24). Quantitative analysis showed 45- and 60-fold increases in the MT message in response to Zn2+ and Cd2+, respectively. Surprisingly, MT expression was dramatically inhibited when the cells were exposed to 100 µM Cr6+ for 15 min before treatment with Zn2+ (100 µM) or Cd2+ (30 µM) for 3 h (Fig. 1B, lanes 5 and 6). Hexavalent chromium alone was unable to induce MT gene expression in HepG2 cells at concentrations ranging from 10 to 100 µM (data not shown). A similar inhibitory effect of Cr6+ on heavy metal-induced MT gene expression was also observed when cells were pretreated with Cr6+ for 15 min and removed before Cd2+ or Zn2+ treatment (data not shown). Because MT-IIA is the major species of MT expressed in human cells, we measured its expression in HepG2 cells by RT-PCR using gene-specific primers. Quantitative analysis of the RT-PCR data revealed an 18-fold increase of the basal MT-IIA message after Zn2+ (100 µM) treatment, whereas Cr6+ (100 and 250 µM) did not change the basal MT-IIA expression in this cell line (Fig. 1C, lanes 24). As observed earlier, Zn2+-induced MT-IIA expression was reduced to basal levels upon exposure of the cells to Cr6+ (Fig. 1C, lanes 2, 5, and 6).
Induction of MT genes in response to heavy metals occurs at the level of transcription (22). To investigate whether Cr6+ affected heavy metal-induced transactivation of MT genes, we performed a nuclear run-on assay with nuclei isolated from HepG2 cells either untreated or treated for 2 h with Zn2+ (100 µM), Cr6+(100 µM), or Cr6+ and Zn2+. The results showed 810-fold increases in MT transcripts in zinc-treated cells compared with control. When the cells were pretreated with Cr6+ for 15 min before the addition of Zn2+, the induction decreased by 23-fold, whereas Cr6+ alone had no effect (Fig. 1D, upper lane). The GAPDH level in each lane was comparable, demonstrating that an equal amount of RNA was used in each sample (Fig. 1D, lower panel). These results demonstrate that the inhibitory effect of Cr6+ on Zn2+-orCd2+-induced expression of MT occurs primarily at the level of transcription.
Hexavalent Chromium Does Not Affect the Expression of Two
MTF1-independent, Cadmium-inducible Genes HSP-70 and Heme Oxygenase 1
(HO-1)We next addressed whether the inhibitory effect of
Cr6+ was due to a global decline in gene expression during the
treatment regimen. We did not observe any inhibitory effect of Cr6+
on housekeeping genes like GAPDH and -actin
(Fig. 1, B and
C). To investigate this issue further, we selected two
other heavy metal-inducible genes, namely, heat shock protein 70
(HSP-70) and heme oxygenase 1 (HO-1). For this purpose,
HepG2 cells were treated for 3 h either with Zn2+ (100
µM)/Cd2+ (30 µM) or Cr6+
(100 µM) alone or in combination. Total RNA isolated from these
cells was subjected to Northern blot analysis with 32P-labeled
HSP-70 and MT-I cDNA. The basal expression of HSP-70 was relatively high in
these cells and was not affected by Zn2+ or Cr6+
(Fig. 2A, upper
panel, lanes 1, 3, and 4). The expression of HSP-70 increased
5-fold after Cd2+ treatment, and it was not blocked significantly
by Cr6+ pretreatment (compare lanes 2 and 6).
However, as shown earlier (Fig.
1), both Cd2+-and Zn2+-induced MT expression
was abolished by the presence of 100 µM Cr6+
(Fig. 2A, middle
panel). Unlike HSP-70, the basal level of hemeoxygenase-1 (HO-1) was
undetectable in HepG2 cells. After treatment of these cells with
Cd2+ the HO-1 level was, however, robustly elevated, whereas
Cr6+ exposure did not alter HO-1 expression
(Fig. 2B, lanes
14). As observed with HSP-70, Cd2+-induced expression
of HO-1 remained unaffected upon exposure to 50 or 100 µM
Cr6+ (Fig.
2B, lanes 5 and 6). These results
clearly demonstrate that the drastic reduction of
Zn2+/Cd2+-mediated MT expression by Cr6+ is
not a global phenomenon.
Overexpression of MTF1 Can Overcome the Inhibitory Effect of Hexavalent Chromium on Heavy Metal-induced Expression of MT GenesInhibition of Zn2+- or Cd2+-induced expression of MT by Cr6+ and not of HSP-70 or HO-1 suggested that the transcription factor MTF1, specific for MT genes, might be a potential target of chromium. MTF1 is essential for both basal and heavy metal-induced expression of the MT genes (30). It was logical to investigate whether overexpression of MTF1 could counteract the inhibitory effect of Cr6+. To address this issue, we used a mouse fibroblast cell line (dko-7), from which both copies of endogenous MTF1 was deleted (23). We generated several stable cell lines (puromycin-resistant) expressing variable levels of FLAG-tagged human MTF1 by transfection with a recombinant retroviral vector (pBabe-hMTF1-FLAG). The level of MTF1 expression in these cell lines was determined by Western blot analysis with anti-FLAG antibodies that detected a specific polypeptide of 110 kDa only in cells expressing recombinant MTF1 (MTF111 and MTF112) but not in the parental dko-7 cells (Fig. 3A, lanes 13). We also measured DNA binding activity in whole cell extracts from these cell lines with a 32P-labeled MRE-s oligonucleotide corresponding to the specific binding site for MTF1 (30). A specific DNA-protein complex could only be detected in MTF1-expressing cell lines but not in dko-7 cells (MTF1 null, Fig. 3B, lanes 13). Quantitation of the 32P signal in the MRE-s·MTF1 complex revealed that MTF1 DNA binding activity in MTF112 cells was 5-fold higher than that of MTF111 cells, which correlated well with the expression level of MTF1 in these cells. The identity of the protein bound to MRE-s as MTF1 was confirmed by competition of the complex with a 100-fold molar excess of unlabeled MRE-s oligo (lanes 46) but not with Sp1 consensus oligo (lanes 79). Also, a supershift of the DNA-protein complex with anti-MTF1 antibody but not with STAT3 antibody (Fig. 3C) confirmed that the complex was indeed MTF1.
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Next we investigated whether the basal and heavy metal-induced MT
expression was higher in MTF112 cells compared with MTF111
cells. As expected, Northern blot analysis showed that both basal and
zinc-induced MT-I expression was significantly (78-fold) higher in
MTF112 cells than that observed in MTF111 cells
(Fig. 4A, lanes
1 and 3 and 5 and 7, and B). We next
investigated whether Cr6+ had a differential effect on MT-I
induction in these cells. As observed in HepG2 cells, Cr6+ alone
could not activate MT-I expression in either cell line
(Fig. 4, A, lanes 2
and 6, and B), but it inhibited zinc-mediated MT-I induction
in both cell lines. The Cr6+-mediated inhibition was more
pronounced in MTF111 cells (80%) than in MTF112 cells
(Fig. 4, A, lanes 4
and 8, and B). These data suggest that MTF1 is indeed one of
the targets of Cr6. Cd2+-induced MT expression was also
inhibited in a dose-dependent manner by Cr6+ in MTF112 cells
(Fig. 4, C and
D). MT mRNA levels increased 65-fold after exposure
to Cd2+ (lane 2) and was reduced to 20-fold and 4-fold by
co-treatment with 100 and 200 µM Cr6+, respectively
(Fig. 4, C, lanes 4
and 5, and D). Higher concentrations of Cr6+ were
needed to achieve significant inhibition of MT-I induction in
MTF112 cells (200 µM versus 100 µM
used in control cells, Fig. 1, A
and B), as these cells contain relatively high levels of
MTF1. These results reinforce the notion that MTF1 protects the
Zn2+- and Cd2+-induced MT expression from the inhibitory
effect of Cr6+.
Hexavalent Chromium Also Inhibits MTF1-dependent Expression of
ZnT-1We next investigated whether Cr6+ could
down-regulate the expression of another MTF1 target gene in response to heavy
metals. The gene for the plasma membrane zinc transporter (ZnT-1)
also harbors an MRE on its proximal promoter, and its transcription is induced
by zinc (23). ZnT-1 exports
Zn2+ from the intracellular pool to maintain zinc homeostasis in
the cell (40). To study the
effect of Cr6+, cell lines expressing a differential level of MTF1
(MTF111 and MTF112) were treated with Zn2+ (100
µM) and/or Cr6+ (100 µM), and total RNA
isolated from these cells was subjected to RT-PCR with mouse ZnT-1-specific
primers. Both cells induced ZnT-1 in response to Zn2+, and the
level of expression was 3-fold higher in MTF112 cells compared with the
MTF111 cells, whereas the -actin level remained unaltered in both
cell lines (Fig. 5, compare
lanes 2 and 6). Like MT-I, ZnT-1 expression was not
up-regulated by treatment with Cr6+ alone (lanes 3 and
7), whereas pretreatment of the cells with Cr6+ resulted
in drastic reduction in the Zn2+-induced ZnT-1 levels in both cell
lines (compare lanes 2 and 4 with lanes 6 and
8). It is noteworthy that the inhibition in MTF112 cells (45%)
was less pronounced than that in MTF111 (72%). These data demonstrate
that Cr6+ down-regulates Zn2+-activated expression of at
least two MTF1 target genes and reemphasizes the inverse correlation between
the MTF1 levels and the inhibitory effect of Cr6+.
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Hexavalent Chromium Does Not Inhibit Zinc-induced Occupancy of MT-IIA and MT-I Promoters in VivoIn response to heavy metals such as Zn2+ or Cd2+, distinct footprints at MREs, MLTF/ARE, and Sp1 binding sites on the MT-I promoter in vivo and concurrent transcriptional activation have been observed (34). MTF1 is activated after treatment with heavy metals and binds to multiple cis elements (MREs) on the MT promoter. Because MTF1 is a transcription factor with six zinc fingers in its DNA binding domain, we reasoned that the inhibitory effect of chromium is probably mediated through inactivation of these fingers. To address whether Cr6+ interferes with Zn2+-induced occupancy of MREs and other cis elements on the MT promoter in the chromatin context, we performed in vivo genomic footprinting (IVGF) analysis. We selected two different cell lines (HepG2 and MTF111) and two promoters (MT-IIA and MT-I) to explore whether the effect of Cr6+ is specific to an MT isoform or to cell type. To analyze in vivo footprinting of the MT-IIA promoter, HepG2 cells were treated with Zn2+ and/or Cr6+ for 1.5 h and exposed to limited DMS treatment, and DNA was prepared (see "Materials and Methods" for details). Human MT-IIA immediate upstream promoter harbors various cis elements including MREs, AP-1, Sp1 and glucocorticoid response element (Fig. 6A). Treatment of HepG2 cells with zinc induced footprinting at G residues encompassing the MRE-f site and the glucocorticoid response element site on the lower strand of the MT-IIA promoter (Fig. 6B). MTF1 harbors six zinc fingers, whereas glucocorticoid receptor has two zinc fingers in its DNA binding domain and are activated by zinc (41), which explains the zinc induced footprinting of these factors on the MT-IIA promoter. There was no apparent footprinting at the AP-2/AP-1 site in control or treated cells. IVGF analysis of the upper strand of the MT-IIA promoter revealed constitutive footprinting at the MRE-a site, where a G residue within the cis element was hypersensitive and three G-residues flanking the MRE-a were protected from DMS-induced methylation (Fig. 6C). There was a slight change in the MRE-a footprinting profile after zinc treatment, when a second hypersensitive G residue was detected in addition to the constitutive one. The MRE-a footprinting in the control cells might be sufficient to explain the basal level of MT-IIA expression observed in the HepG2 cells (Fig. 1C). We also observed zinc-induced footprinting at the composite Sp1/MRE-b site, where three consecutive G-residues were protected and one G-residue was hypersensitive (Fig. 6C). All the footprints detected on MT-IIA promoter in control and zinc-treated cells remained unaltered when cells were treated with Cr6+ alone or before exposure to Zn2+. None of the zinc-induced footprints detected in the upper or lower strand of the MT-IIA promoter were observed in the Cr6+ treated cells, where the G-ladder remained identical to control DNA. These results also rule out occupancy of a negative element in the immediate promoter region by a potential repressor upon chromium exposure. To our knowledge this is the first IVGF study of human MT-IIA promoter, which revealed zinc-inducible occupancy of not only MREs but also glucocorticoid response element. We also measured DNA binding activity of MTF1 in the nuclear extract prepared from HepG2 cells treated with zinc, chromium, or both. The results showed that zinc-induced activation of MTF1 was not blocked by pretreatment with chromium, which itself could not activate MTF1 binding (data not shown). These results clearly demonstrate that neither translocation of MTF1 to the nucleus nor its binding to the promoter in response to zinc is compromised by chromium pretreatment.
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To analyze in vivo footprinting of the mouse MT-I promoter (Fig. 6D) we subjected MTF111 cells to the same treatment regimen as described for HepG2 cells. Treatment with Zn2+ induced distinct footprinting at MRE-a, -b, -c, and -d sites on the lower strand in MTF111 cells. Some of the G residues at the MRE-a, -b, -c', and -d sites were protected, and some at the MRE-c' site were hypersensitive to DMS treatment due to binding of a transcription factor (Fig. 6E). Zn2+-induced footprinting was also observed at MRE-c' and MRE-e in the upper strand of the MT-I promoter (Fig. 6F). In untreated MTF111 cells, the upper strand of the MT-I promoter was occupied at the MLTF/ARE, Sp1, and MRE-d/Sp1 binding sites. All the constitutive footprints observed in the control cells remained unaffected in the Cr6+-treated cells. No inducible footprints appeared in the Cr6+-treated cells, as observed in Zn2+-exposed cells (Fig. 6, E and F, lanes 2 and 4). The footprinting profiles of the MT-I promoter in cells treated with Zn2+ and Cr6+ were identical to that in response to Zn2+ alone (Fig. 6, D and F, lanes 3 and 5). Zn2+-induced occupancy of MREs and other sites remained intact, and no novel footprinting appeared in either the MT-I or MT-IIA promoter after Cr6+ treatment. These data confirmed that Cr6+ did not alter the interaction of transacting factors (basal or inducible) with cognate cis elements on the MT-I or MT-IIA promoters.
Transient Overexpression of MTF1 Can Alleviate the Inhibitory Effect of Cr6+ on Zn2+- and Cd2+-induced MT-I Promoter ActivityIf Cr6+ affects the transactivation potential of MTF1, transient overexpression of MTF1 should alleviate the inhibitory effect of Cr6+ on MT-I promoter activity in transfection studies. To test this possibility, HepG2 cells were transiently transfected with a MT-I promoter/luciferase reporter plasmid (pMT-Luc) (38) and treated with Zn2+, Cd2+, and/or different concentrations of Cr6+. Both Zn2+ and Cd2+ activated the MT-I promoter when the cells were treated with the heavy metals for 3 h before harvest. Cr6+ inhibited the basal promoter activity by 40 and 75% at a concentration of 50 and 100 µM, respectively, whereas 10 µM Cr6+ had no detectable effect (Fig. 7A). Both the Zn2+- and Cd2+-induced promoter activities were inhibited significantly by 100 µM Cr6+ (65 and 70%, respectively). To determine the effect of MTF1 overexpression, HepG2 cells were next co-transfected with pMT-Luc with pRL-TK as an internal control along with different amounts of the human MTF1 expression vector (pchMTF1). As observed earlier (38), the basal MT-I promoter activity increased with increasing amounts of cotransfected hMTF1 expression vector (Fig. 7B, lanes 1, 4, 7, and 10). The addition of Zn2+ further enhanced this promoter activity, although the differences between the basal and Zn2+-induced activity diminished with increased expression of hMTF1 (lanes 2, 5, 8, and 11). The addition of Cr6+ (100 µM) before Zn2+ inhibited the promoter activity significantly (lanes 3, 6, 9, and 12). This could be reversed by overexpression of MTF1 protein. In the absence of MTF1 overexpression Zn2+-induced MT-I promoter activity was inhibited by 71% in the presence of Cr6+ (Fig. 7B, lanes 2 and 3). The inhibition decreased to 65% when 6 µg of pchMTF1 was cotransfected in HepG2 cells with pMT-Luc (lanes 5 and 6). This recovery from the Cr6+-induced effect was even more pronounced when 8 µg (lanes 8 and 9) and 10 µg (lanes 11 and 12) of the pchMTF1 were cotransfected (45 and 20% inhibition, respectively). These data further confirm the notion that MTF1 overexpression indeed protects MT-I promoter activity from the inhibitory effect of Cr6+.
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Hexavalent Chromium Inhibits the Function of all Three Transactivation Domains of MTF1IVGF analysis and electrophoretic mobility shift assay (data not shown) clearly demonstrated that the DNA binding activity of MTF1 was not compromised in cells treated with Cr6+. We therefore hypothesized that the transactivation domains of MTF1 could possibly be the targets of Cr6+. MTF1 has three different transactivation domains, namely acidic domain (Ac), the proline-rich domain (P), and the serine-threonine rich domain (ST) (Fig. 8). A complex interaction between these domains is required for MTF1 activity (21). To explore which functional domain of MTF1 is the target of Cr6+, we used fusion proteins consisting of the DNA binding domain of the heterologous yeast factor Gal4 and different regions of the mouse MTF1 protein (21). HepG2 cells were cotransfected with a luciferase reporter plasmid that contained five Gal4 binding sites on the promoter (pG5-Luc, Promega) and different Gal4/MTF1 fusion proteins. The Gal4 fusion proteins used in this experiment are [Gal4]Ac/P/ST, [Gal4]Ac/P, [Gal4]Ac, [Gal4]ST, and [Gal4]P. The cells were treated with Zn2+ and/or Cr6+ 3 before harvest. The expression of [Gal4]Ac/P/ST and [Gal4]Ac resulted in similar basal pG5-Luc promoter activity, which remained comparable after Zn2+ treatment (Fig. 8B, lanes 1 and 9, and 3 and 11). This insensitivity to Zn2+ can be attributed to the lack of a zinc finger domain in the MTF1 fusion proteins, as the rest of the domains responded minimally to Zn2+ (21). When the [Gal4]ST fusion protein was overexpressed, pG5-Luc activity was decreased to 30% that observed in presence of [Gal4]Ac/P/ST (compare lanes 5 and 7 to lanes 1 and 3), whereas [Gal4]Ac/P and [Gal4]P showed less than 10% of the activity (compare lanes 13, 15 and 17, 19 to lanes 1 and 3). When exposed to Cr6+, cells expressing the five different fusion proteins exhibited on average a 6070% reduction in the activity of the pG5-Luc promoter. A similar degree of inhibition was observed irrespective of the presence or absence of Zn2+. These results suggest that the functions of all three transactivation domains of MTF1 are susceptible to the inhibitory effect of Cr6+.
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Hexavalent Chromium Can Enhance Zinc and Cadmium-induced Apoptosis, Which Can Be Counteracted by MTF1 OverexpressionExposure of cells in culture to heavy metals such as Cr6+ (14), Cd2+ (42), and Zn2+ (43) induces apoptosis, particularly at an early stage, which is eventually followed by necrosis if the heavy metals are not scavenged by detoxifying proteins like metallothioneins. Because Cr6+ inhibits the heavy metal-induced MT expression and increased MTF1 expression counteracted the inhibitory effect, it was of interest to investigate how MTF1 null cells respond to heavy metal-induced apoptosis in comparison to overexpressing cells. For this purpose, we first used annexin V staining to analyze the onset of apoptosis under different treatment conditions (see "Materials and Methods" for details) using dko-7 (MTF1 null) and MTF112 cells (MTF1-overexpressing) (Fig. 3). The cells were either left untreated (control) or treated with 100 µM Zn2+, 100 µM Cr6+, or 100 µM each Zn2+/Cr6+ for 2 h followed by annexin V staining for 15 min. The cells were counter-stained with PI. Treatment with Zn2+ and/or Cr6+ for a short time period (2 h) induced apoptosis in dko-7 cells (MTF null) under all three treatment regimens (Fig. 9, panel A). Phosphatidylserine residues that are translocated from the inner to the outer layer of the plasma membrane at an early stage of apoptosis after membrane damage are detected by annexin V binding. As apoptosis progresses, the membrane loses its integrity, and a halo of green stain appears within the entire cell. Precise analysis and comparison of the fluorescent profile showed that the entire cell was stained green when dko-7 cells were exposed to Zn2+ alone or along with Cr6+ (Fig. 9A, 2a and 4a). In the Cr6+-treated cells, green specks on the plasma membrane were distinctly visible with concurrent staining of the cytoplasm (Fig. 9A, 3a). This observation suggested that MTF1 null cells offered minimum resistance to heavy metal-induced apoptosis. When MTF112 cells were subjected to identical treatment, annexin V-FITC staining was barely detectable in cells treated for 2 h with Zn2+ or Cr6+ alone (Fig. 9B, 2a and 3a). However, simultaneous treatment of MTF112 cells with both heavy metals induced onset of apoptotic cell death, as was evident from the appearance of green specks on the membrane periphery (Fig. 9B, 4a). The most logical explanation for this observation is that MTF112 cells resist Zn2+-induced apoptosis by inducing MTs to scavenge intracellular Zn2+ and by inducing ZnT-1 that effluxes excess intracellular Zn2+. The ability of MTF112 cells to resist Cr6+-induced apoptosis is likely due to the higher basal levels of MT-1 and ZnT1 expression in these cells (Figs. 4 and 5). dko-7 cells that lack MTF1 cannot express these proteins and are prone to apoptosis when challenged with metals. Although MTF112 cells could resist zinc treatment for at least 2 h, exposure to Cr6+ before zinc treatment augmented the toxic effect of zinc and led to onset of apoptosis. This deleterious effect of Zn2+ and Cr6+ on MTF112 cells can be attributed to decreased expression of MT and ZnT-1 in the presence of Cr6+ (Figs. 4 and 5). Strong PI staining was visible in metal-treated dko-7 cells (Fig. 9A, 2b4b). Because the dye can only penetrate cells where the membrane integrity is lost, it can be assumed that apoptosis is prevalent in these cells. Minimal PI staining in MTF112 cells implicates resistance of these cells toward these heavy metals under the experimental conditions used in this study.
We also analyzed the induction of apoptotic cell death in dko-7 and MTF112 cells by heavy metals using the TUNEL assay. This assay detects cellular endonuclease-mediated ordered DNA fragmentation, a late event in apoptotic cell death. Both dko-7 and MTF112 cells were treated with Zn2+ and/or Cr6+ for 2 h and the TdT (terminal deoxynucleotidyltransferase) assay was performed following the manufacturer's protocol. Insignificant levels of FITC staining were observed in the untreated dko-7 cells, whereas in cells treated with heavy metals alone or in combination (this is the same treatment done for annexin staining) TUNEL-positive FITC staining was observed (Fig. 10, panel A). On the other hand, when MTF112 cells were treated with Zn2+, Cd2+, or Cr6+, TUNEL-positive cells were not detected irrespective of the treatment condition (Fig. 10, panel B). This observation supports the data from the annexin V assay, where onset of apoptosis in MTF112 cells was not observed in the presence of Cr6+ or Zn2+ alone. Unlike dko-7 cells TUNEL-positive cells were not detected in MTF112 cells exposed to Cr6+ before Zn2+ or Cd2+ treatment. We have seen earlier (by annexin V staining) that a combination of Zn2+ and Cr6+ in 2 h can only lead to the onset of apoptosis in MTF112 cells. Because TUNEL detects a late event in the apoptosis, we did not expect to detect significant TUNEL-positive staining in MTF112 cells under this treatment regimen. Positive PI staining was observed in both dko-7 and MTF112 cells, as cells were permeabilized before performing TUNEL assay. This set of data suggests that Cr6+ augments the toxic effect of heavy metals such as Zn2+ and Cd2+. Also, MTF1 null cells (dko-7) are more vulnerable to heavy metal-induced apoptotic cell death and increased expression of MTF1 can protect the cells from a heavy metal insult.
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DISCUSSION |
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It should be emphasized that the inhibitory effect of Cr6+ on
heavy metal-induced MT gene expression is not merely a result of
global toxicity imposed by Cr6+, as Cd2+-induced
expression of stress response genes, namely HSP-70 and HO-1,
remained unaltered under this condition. We have also observed unabated
expression of housekeeping genes such as GAPDH and
-actin under the same treatment regimen. It should also be
noted that at a concentration similar to that of Zn2+ (100
µM), Cr6+ could block zinc-induced MT expression in
HepG2 cells. Our study revealed that the transactivation potential, but not
the DNA binding activity, of the key transcription factor MTF1 is the target
of Cr6+ inhibition. The specificity of this effect was evident from
the protection afforded by overexpressed MTF1 on the hexavalent
chromium-mediated down-regulation of ZnT-1, another MTF1-responsive
gene. In this context, it is noteworthy that unlike Cd2+ or
As3+, Cr6+ inhibits the expression of dioxin-inducible
genes, probably by interfering with the transactivation potential of the
aromatic hydrocarbon receptor (AHR) rather than inhibiting its DNA binding
activity (46). One intriguing
question is which ionic form of chromium interferes with MT gene activation by
other heavy metals. Electron spin resonance study has shown that hexavalent
chromium is spontaneously reduced to pentavalent chromium immediately after
uptake (8) and then gradually
reduced to the stable, relatively less toxic, Cr3+ form. One of the
three ionic forms of Cr, namely (V), (IV), and (III), might be the active form
that antagonizes MT induction. It was not possible to identify the ionic form
of inhibitory chromium in the present study due to unavailability of soluble,
membrane-permeable Cr(V) and Cr(IV) compounds.
It is noteworthy that Cr6+ preferentially suppresses the expression of genes that exhibit a heavy metal-mediated increase in expression via an MTF1 binding site on their promoter. Such genes include MT-I, MT-IIA, and ZnT-1. The expression of other heavy metal-inducible genes such as HSP-70 and HO-1 continued unabated in the presence of Cr6+. The lack of an MTF1 consensus element (MRE) on the promoters of the latter two genes further confirms that MTF1 is indeed the target of Cr6+ inhibition. Sp1 binding site is another cis element that is occupied on both the MT-I and MT-IIA promoter with or without heavy metal treatment. IVGF analysis of the MT-I promoter in MTF111 cells in the presence of Zn2+ and/or Cr6+ showed no alteration in factor binding at this site, suggesting that Sp1 DNA binding was not affected by Cr6+. Similar Zn2+-inducible footprinting at the Sp1/MRE-b composite site on the MT-IIA promoter remained unchanged when HepG2 cells were treated with Cr6+ before zinc exposure. That the transactivation potential of Sp1 is not a target of Cr6+ inhibition is further substantiated by the observation that Cr6+ does not affect the basal or Cd2+-induced expression of HSP-70 or HO-1. It is known that Sp1 either alone or in cooperation with other transactivators regulates expression of both HSP-70 and HO-1 gene (47, 48). A recent study has shown that Zn2+ also induces translocation of recombinant MTF1-GFP from the cytoplasm to the nucleus (49). Because the Zn2+-induced IVGF profiles of the MT-I and MT-IIA promoters are not affected by Cr6 and the activation of MTF1 in the nuclear extracts of cells treated with both metals remains unaltered, it is safe to conclude that Cr6+ does not inhibit nuclear translocation of MTF1.
It has been hypothesized that Cd2+ and other heavy metals act by releasing Zn2+ from intracellular zinc storage proteins, leading to activation of MTF1 (22). Because in a wide range of concentrations Cr6 + (10250 µM, Fig. 1B, and some data not shown) could not activate the MT promoter, one can speculate that unlike other heavy metals, it fails to mobilize Zn2+ from the intracellular storage sites. However, this notion does not explain why Cr6+ blocks activation of MT expression in response to other metals. The DNA binding activity of MTF1, a Zn2+-mediated process is not compromised in cells treated with Zn2+ and Cr6+. Indeed, the data presented here point to an inhibitory effect at the transactivation stage subsequent to DNA binding by MTF1. This conclusion was supported further by the observation that stable or transient overexpression of MTF1 in mouse MTF1 null cells and HepG2 cells can significantly protect the ability of these cells to respond to Zn2+ or Cd2+ by expressing the MT gene. The extent of this protection correlates directly with the level of MTF1 expression. Because there is no dramatic increase in MTF1 transcript levels in response to heavy metals that normally leads to robust transcription of the MT genes, MTF1 probably undergoes significant post-translational modification in this process (26, 50). We have not, however, detected any significant change in MTF1 phosphorylation/dephosphorylation when MTF112 cells were treated with either Cr6+ alone or in combination with Zn2+ or Cd2+ (data not shown).
The present study has also shown that Cr6+ interferes with the function of all three different activation domains of MTF1 in concert. This finding is consistent with an earlier observation (21) that demonstrated the role of all three domains namely, N-terminal, C-terminal, and middle domains, of MTF1 in the activation of the MT-I promoter. Cr6+ may inhibit interaction between the MTF1 transactivation domains with a coactivator(s) or other general transcription factor by an as yet unknown mechanism. Identification of co-activators or general transcription factors with which MTF1 interacts is a big challenge to the field and is beyond the scope of the present study.
Both Zn2+- and Cd2+-induced apoptosis was observed at an early stage in MTF1 null cells (dko-7), whereas MTF1-overexpressing cells (MTF112) were resistant to these metals. It has been reported that zinc can be internalized through the mitochondrial uniport, leading to generation of reactive oxygen species and induction of apoptosis (51). Unlike MTF112 cells, the lack of MT-I and ZnT-1 expression in dko-7 cells would result in increased accumulation of free intracellular zinc, facilitating the cell death process. From all the data gathered so far it is obvious that pretreatment with Cr6+ would augment the toxic effect. This has been nicely demonstrated in MTF112 cells, where a combination of Zn2+ and Cr6+ resulted in annexin V-sensitive membrane disintegration, whereas the cells treated with Zn2+ alone showed no sign of apoptosis. From this data it is evident that suppression of MT and ZnT1 expression in the presence of Cr6+ led to an imbalance in the intracellular zinc pool, resulting in onset of apoptosis. However, the observed apoptosis in MTF112 cells showed significant delay compared with dko-7 cells. This observation reemphasizes the role of MT-I and ZnT-1 in scavenging toxic heavy metals and, consequently, the need for functional MTF1, required for induction of the above proteins.
It is important to realize that the effects of MTF1 dysfunction on the
cellular process caused by Cr6+ are multifaceted. Uninterrupted
expression of the zinc transporters as well as the zinc-metallothionein
storage proteins is essential for the maintenance of Zn2+
homeostasis in cells. Of the four zinc transporters, ZnT-1 is the only one
present on the plasma membrane, where it functions as a zinc effluxor
(40). ZnT-1 is
expressed ubiquitously, and the homozygous knockout is embryonic-lethal
(52). When the expression of
MT-I/MT-II and ZnT-1 are impaired in the presence of Cr6+, the
capacity of the cells to maintain Zn2+ homeostasis is severely
impeded. Our previous study demonstrates that the MT-I gene is highly
induced by heavy metals and probably plays a protective role when mice are
exposed to restrained stress
(53) or viral infection
(28). It is, therefore,
conceivable that the ability to cope with infection and stress where zinc
plays a protective role will be hindered upon exposure to Cr6+. The
liver-enriched transcription factor C/EBP is also a candidate target
gene for MTF1 (24). This gene
is important for cellular stress response
(54,
55) and proper liver
development (56). It is
logical to speculate that the overall capacity of cells to respond to stress
will be diminished in the presence of Cr6+. Because the
C/EBP
family of proteins is critical for liver development, it would be
of interest to explore the role of Cr6+ in mammalian development.
Future studies will address this and related issues.
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FOOTNOTES |
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These authors contributed equally to this work.
¶
To whom correspondence should be addressed: Dept. of
Molecular and Cellular Biochemistry, The Ohio State University, College of
Medicine, 333 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.:
614-688-5494; Fax: 614-688-5600; E-mail:
ghoshal.1{at}osu.edu.¶
To whom correspondence should be addressed: Dept. of Molecular and Cellular
Biochemistry, The Ohio State University, College of Medicine, 333 Hamilton
Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-688-5494; Fax:
614-688-5600; E-mail:
jacob.42{at}osu.edu.
1 The abbreviations used are: MT, metallothionein; ZnT-1, zinc transporter 1;
HSP-70, heat shock protein 70; HO-1, heme oxygenase 1; MRE, metal-responsive
element; MTF1, metal-responsive transcription factor 1; TUNEL, terminal
deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labeling; IVGF,
in vivo genomic footprinting; PI, propidium iodide; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; LM-PCR,
ligation-mediated PCR; FITC, fluorescein isothiocyanate; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMS, dimethyl
sulfate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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