Down-Regulation of the Expression of the FIH1 and ARD1 Genes at the Transcriptional Level by Nickel and Cobalt in the Human Lung Adenocarcinoma A549 Cell Line

Abstract

Although nickel and cobalt compounds have been known to cause induction of the transcription factor hypoxia-inducible factor 1 (HIF-1) and activation of a battery of hypoxia-inducible genes in the cell, the molecular mechanisms of this induction remain unclear. The post-translational modification of HIF-1a, the oxygen-sensitive subunit of HIF-1, regulates stabilization, nuclear translocation, DNA binding activity, and transcriptional activity of the protein. Among the enzymes regulating the post-translational modification of HIF-1a, the factor inhibiting HIF-1 (FIH-1) hydroxylates the protein at asparagine 803, suppressing the interaction of HIF-1a with transcription coactivators p300/CBP and reducing the transcriptional activity of the protein. ARD-1, the acetyltransferase, acetylates HIF-1a at lysine 532, which enhances the interaction of HIF-1a with pVHL. Therefore, FIH-1 and ARD-1 negatively regulate the transcriptional activity and the stability of HIF-1a. We examined the mRNA levels of FIH-1 and ARD-1 genes after exposure nickel (II) or cobalt (II) to the cell and found that both genes were down-regulated by the chemical treatment, which may lead to reduced levels of both proteins and result in increased level of HIF-1a and its transcriptional activity.

Full text

Int. J. Environ. Res. Public Health 2005, 2(1), 10–13
International Journal of
Environmental Research and Public Health
ISSN 1660-4601
www.ijerph.org
© 2005 by MDPI
© 2005 MDPI. All rights reserved.
Down-Regulation of the Expression of the FIH-1 and ARD-1 Genes at
the Transcriptional Level by Nickel and Cobalt in the Human Lung
Adenocarcinoma A549 Cell Line
Qingdong Ke
1
, Thomas Kluz
1
, and Max Costa
1*
1
Nelson Institute of Environmental Medicine, New York University, School of Medicine, 57 Old Forge Road, Tuxedo,
New York 10987, USA
*Correspondence to Dr. Max Costa. E-mail: Costam01@nyu.edu
Received: 15 November 2004 / Accepted: 06 February 2005 / Published: 30 April 2005
Abstract: Although nickel and cobalt compounds have been known to cause induction of the transcription factor
hypoxia-inducible factor 1 (HIF-1) and activation of a battery of hypoxia-inducible genes in the cell, the
molecular mechanisms of this induction remain unclear. The post-translational modification of HIF-1a, the
oxygen-sensitive subunit of HIF-1, regulates stabilization, nuclear translocation, DNA binding activity, and
transcriptional activity of the protein. Among the enzymes regulating the post-translational modification of HIF-
1a, the factor inhibiting HIF-1 (FIH-1) hydroxylates the protein at asparagine 803, suppressing the interaction of
HIF-1a with transcription coactivators p300/CBP and reducing the transcriptional activity of the protein. ARD-1,
the acetyltransferase, acetylates HIF-1a at lysine 532, which enhances the interaction of HIF-1a with pVHL.
Therefore, FIH-1 and ARD-1 negatively regulate the transcriptional activity and the stability of HIF-1a. We
examined the mRNA levels of FIH-1 and ARD-1 genes after exposure nickel (II) or cobalt (II) to the cell and
found that both genes were down-regulated by the chemical treatment, which may lead to reduced levels of both
proteins and result in increased level of HIF-1a and its transcriptional activity.
Keywords: nickel, cobalt, hypoxia, HIF-1a, FIH-1, ARD-1
Introduction
According to the International Agency for Research
on Cancer (IARC 1990), both soluble and insoluble
nickel compounds have long been established as human
and animal carcinogens [1]. And cobalt compounds are
carcinogenic in animals [2]. Epidemiological studies
have shown that environmental or occupational exposure
to nickel or cobalt compounds could cause lung and
nasal cancers, asthma, fibrosis, pneumonitis and some
other lung injuries [1-5]. Despite the various differences
among the compounds of these two metals regarding the
biochemical and molecular mechanisms of their toxicity
and carcinogenicity, they mimic hypoxia to induce the
HIF-1a transcription factor and hypoxia-inducible genes
[5, 6], which are believed to play important roles in
carcinogenesis. However, the complete mechanisms by
which nickel and cobalt compounds induce HIF-1a are
still unknown, although several sites of their impact on
HIF-1a have been described [7-9].
The transcription factor hypoxia-inducible factor 1
(HIF-1) plays an essential role in cellular oxygen
homeostasis [10, 11]. HIF-1 is a heterodimeric complex
composed of alpha and beta two subunits [12]. The beta
subunit is also the heterodimerization partner for the aryl
hydrocarbon receptor (AhR) and thereby called aryl
hydrocarbon receptor nuclear translocator (ARNT) that
is constitutively expressed; whereas the alpha subunit
(HIF-1a) is highly oxygen-sensitive and is rarely
detectable under normal oxygen tension but is
dramatically induced with hypoxia [12]. Under reduced
oxygen tension, HIF-1a is stabilized and translocates to
the nucleus, where it dimerizes with ARNT. Then the
active HIF-1 stimulates the transcription of genes
involved in angiogenesis, cell survival, glucose transport
and metabolism [13, 14].
HIF-1a is regulated by a reduced oxygen level largely
at its post-translational modifications, resulting in
stabilization, nuclear translocation, DNA binding
activity, and transcriptional activity of the protein. The

Int. J. Environ. Res. Public Health 2005, 2(1)
11
post-translational modifications of HIF-1a include prolyl
hydroxylation at proline 402 and 564 within the oxygen-
dependent degradation (ODD) domain by HIF-prolyl
hydroxylases (HPHs) [9, 15-17], asparaginyl hydroxylation
at asparagine 803 in the C-terminal activation domain
(C-TAD) by factor inhibiting HIF-1 (FIH-1) [8, 18, 19],
acetylation of lysine 532 in the ODD domain by an
acetyltransferase ARD-1[20], phosphorylation induced
by p42/p44 mitogen-activated protein kinase (MAPK)
activity [21], as well as ubiquitination by the von Hippel-
lindau (pVHL) complex [22-25].
In normoxia, proline 402 and 564 of HIF-1a are
hydroxylated, which is required for the binding of pVHL
complex and leads to the ubiquitination of the protein,
resulting in targeting of HIF-1a for proteasomal
degradation [15-17, 24]. The interaction of HIF-1a with
pVHL is enhanced by ARD-1-mediated acetylation at
lysine 532 [20]. The acetylation of this lysine residue by
ARD-1 is critical to the proteasomal degradation of HIF-
1a since a mutant with arginine substituting lysine 532
shown no acetylation by ARD-1 was stabilized and had a
decreased interaction with pVHL [20]. At the same time,
hydroxylation of asparagine 803 during normoxia
suppresses interaction of HIF-1a CAD with transcription
coactivators p300/CBP and reduces the transcriptional
activity of the protein [18, 26]. In addition to interacting
with HIF-1a, the asparaginyl hydroxylase FIH-1 also
interacts with pVHL, allowing the formation of
complexes containing HIF-1a, FIH-1, and pVHL [27].
In hypoxia, decreased level of prolyl hydroxylation
due to the limiting oxygen prevents pVHL binding to
HIF-1a, resulting in rapid accumulation of HIF-1a
protein [15, 16, 24]. The acetylation level of HIF-1
gradually decreases as the length of hypoxic exposure
time increases, which is due to the reduced expression of
ARD-1[20]. Meanwhile, stabilized HIF-1a protein is
able to bind to p300/CBP to execute its transcriptional
activity due to decreased level of hydroxylation at
asparagine 803[18, 19]. In addition, during hypoxia,
p42/p44 MAPK activity induces phosphorylation of
HIF-1a and promotes its transcriptional activity [21]. As
a consequence, HIF-1a accumulates and promotes
hypoxic tolerance by activating gene transcription.
Among the enzymes that regulate HIF-1a, prolyl
hydroxylases (HPHs) and asparaginyl hydroxylase (FIH-1)
belong to the family of 2-oxoglutarate-dependent
dioxygenases and require Fe2+, 2-oxoglutarate, O2, and
ascorbate for their reactions [8, 15, 16]. ARD-1
acetyltransferase acetylates HIF-1a by transferring an acetyl
group from acetyl-CoA [20].
Nickel (II) and cobalt (II) exposure in the presence of
oxygen causes accumulation of HIF-1a protein and
induction of its transcriptional activity [28-30], in part
resulting from the inability of HIF-1a binding to pVHL
due to the inhibition of HIF-1a hydroxylation by the
metals [24, 31]. Furthermore, Cobalt (II) has been shown
to inhibit activities of recombinant asparaginyl and
prolyl hydroxylase in vitro [8, 9].
In this study, we have examined the effects of nickel
(II) and cobalt (II) on the gene expression of FIH-1 and
ARD-1 using reverse transcriptase PCR (RT-PCR). Both
genes were down-regulated in the metal-exposed cells,
which might lead to reduced level of both proteins and
result in increased level of HIF-1a and its transcriptional
activity.
Materials and Methods
Cell Culture
Human lung adenocarcinoma A549 cells (CCL185)
were purchased from American Type Culture Collection
(Manassas, VA). Cells were maintained in F-12K
medium (Life
Technologies, Inc., Gaithersburg, MD)
supplemented with 10% fetal
bovine serum and 1%
penicillin/streptomycin (equivalent to 100 units/ml
and
100 µg/ml, respectively) at 37°C as monolayers
in a
humidified atmosphere containing 5% CO
2
.
Chemicals and Cell Exposure
Nickel chloride hexahydrate and cobalt chloride
hexahydrate were purchased from Sigma (St. Louis,
MO). Cells were seeded into 100-mm dishes and
allowed to attach overnight. When cells reached 70–80%
confluence, nickel chloride hexahydrate (0.5 mM,
1.0mM), or cobalt chloride hexahydrate (0.2mM, 0.4
mM) was added to the medium for 24h.
RNA Extraction and RT-PCR
At the end of the treatment, total RNA was isolated
from the cell using the Trizol reagent (Invitrogen,
Carlsbad, CA). The mRNA was isolated using Oligotex
kit (Qiagen, Germany). Reverse transcription was carried
out with SuperScript first-Strand Synthesis System for
RT–PCR (Invitrogen) and 1 µg of mRNA was used for
first-strand cDNA synthesis according to the
manufacturer’s protocol. The PCR was carried out in a
total volume of 50µl and 1µl of first-strand cDNA was
used for amplifying genes. The primers used for the
PCRs were: FIH-1 forward primer,
5'-
GCCAGCACCCACAAGTTCTT-3'; FIH-1 reverse primer,
5'-CCTGTTGGACCTCGGCTTAA-3' ARD-1 forward
primer, 5’-TGGGGTGAGGAGGGGATGG-3’; ARD-1
reverse primer, 5’-GGGAAGATTGTGGGGTATG-3’.
Primers for amplifying GAPDH gene were purchased from
BD Biosciences Clontech. Amplification conditions for
FIH-1 and ARD-1 genes were 95°C for 2 min, 22 cycles
at 95°C for 45 s, 57°C for 45 s, 72°C for 1 min, and
72°C for 5 min; the same for GAPDH gene except that
16 cycles were performed. PCR products were then
resolved on 1.5% agarose gels containing Ethidium
bromide.
Results
Nickel (II) and Cobalt (II) Down-Regulate the Gene
Expression of FIH-1and ARD-1 Genes
The investigation on the mechanisms of the induction
of HIF-1a by nickel (II) and cobalt (II) has been focused
on the effects of the metals on the enzyme activities of
HPHs and FIH-1. Besides the possible effects of the
metals on the enzyme activities, nickel (II) and cobalt
(II) might affect the gene expression of the enzymes, so

Int. J. Environ. Res. Public Health 2005, 2(1)
12
that the levels of the enzymes in the cell could be
affected. Therefore, we examined mRNA levels of FIH-
1 and ARD-1 by exposure A549 cells to nickel (II) and
cobalt (II). As shown in figure 1, nickel (II) and cobalt
(II) decreased FIH-1 and ARD-1 mRNA levels in a
dose-dependent manner. GAPDH gene served as an
internal control. The house keep gene 60S acidic
ribosomal protein has also been used for measuring the
loading control, which gave the same pattern as that of
GAPDH gene (data not shown).
Figure 1: Nickel (II) and Cobalt (II) down-regulate
mRNA levels of FIH-1 and ARD-1genes in A549 cells
A549 cells were treated with NiCl
2
·6H
2
O (0.5mM and 1
mM) or CoCl
2
·6H
2
O (0.2 mM and 0.4mM) for 24 h. The
cells were harvested at the end of the treatment and the
mRNA was isolated. The FIH-1, ARD-1, and GAPDH
genes were amplified by RT-PCR.
Discussion
Nickel (II) and cobalt (II) have long been known to
induce hypoxia-like stress by activating HIF-1a [28-30].
However, the mechanisms of this induction are still
unclear, although several studies have shown effects at
sites of HIF-1a regulation, particularly focusing on the
effects of the metals on the hydroxylases activities[7-9].
Since the HIF-1a hydroxylases require iron (II) as a co-
factor for their activities and iron, cobalt, and nickel are
adjacent in the transition metal group, it has been
suggested that nickel (II) and cobalt (II) may substitute
for the iron (II) in the hydroxylases, thereby, causing the
loss of the enzymatic activity[7]. Unfortunately, there is
no direct evidence to support this hypothesis. Recently,
the cellular ascorbate, another co-factor required for the
hydroxylases activities, has been shown to be depleted
by both metals. Since the role of ascorbate is maintaining
iron in its reduced state (iron II), this depletion may
favor enzyme-bound iron oxidation, which may lead to
the inactivation of the hydroxylases[31].
Besides their effects, direct or indirect, on the
hydroxylases activities, nickel (II) and cobalt (II) are
very likely to induce HIF-1a at additional sites. We
demonstrated here that the mRNA levels of FIH-1 and
ARD-1 genes were down-regulated by both metals,
which could result in reduced levels of the protein
products of these genes. Since both FIH-1 and ARD-1
proteins negatively regulate HIF-1a, decreasing of them
would lead to accumulation of HIF-1a and increase of its
transcriptional activity.
Furthermore, we suspect that nickel (II) or cobalt (II)
may affect the acetylation of HIF-1a not only by down-
regulating the acetyltransferase ARD-1, but also by
decreasing the level of the cellular acetyl-CoA, the
supply of the acetyl group, by the way of shutting down
the Kreb cycle as well as increasing the ratio of
NAD/NADH thereby causing the deacetylation of
histones. Since both nickel and cobalt have previously
been shown to inhibit histone acetylation and increase
the methylation of histone H3 at lysine 9, this effect may
play a role in the down-regulation of these two important
genes which impact negatively upon HIF-1a activation
(Costa et al. Mutation Res. in press).
Our study revealed a possible new mechanism of the
nickel- and cobalt- induction of hypoxia-like stress in the
cell, contributing insight to better understanding of their
carcinogenesis.
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