Cadmium induces transcription independently of intracellular calcium mobilization.
ABSTRACT Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+)](i)) and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal.
In the present report, the effects of cadmium on [Ca(2+)](i) under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+)](i) mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression.
These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription.
- [Show abstract] [Hide abstract]
ABSTRACT: Cellular stress elicited by the toxic metal Cd(2+) does not coerce the cell into committing to die from the onset. Rather, detoxification and adaptive processes are triggered concurrently, allowing survival until normal function is restored. With high Cd(2+), death pathways predominate. However, if sublethal stress levels affect cells for prolonged periods, as in chronic low Cd(2+) exposure, adaptive and survival mechanisms may deregulate, such that tumorigenesis ensues. Hence, death and malignancy are the two ends of a continuum of cellular responses to Cd(2+), determined by magnitude and duration of Cd(2+) stress. Signaling cascades are the key factors affecting cellular reactions to Cd(2+). This review critically surveys recent literature to outline major features of death and survival signaling pathways as well as their activation, interactions and cross talk in cells exposed to Cd(2+). Under physiological conditions, receptor activation generates 2nd messengers, which are short-lived and act specifically on effectors through their spatial and temporal dynamics to transiently alter effector activity. Cd(2+) recruits physiological 2nd messenger systems, in particular Ca(2+) and reactive oxygen species (ROS), which control key Ca(2+)- and redox-sensitive molecular switches dictating cell function and fate. Severe ROS/Ca(2+) signals activate cell death effectors (ceramides, ASK1-JNK/p38, calpains, caspases) and/or cause irreversible damage to vital organelles, such as mitochondria and endoplasmic reticulum (ER), whereas low localized ROS/Ca(2+) levels act as 2nd messengers promoting cellular adaptation and survival through signal transduction (ERK1/2, PI3K/Akt-PKB) and transcriptional regulators (Ref1-Nrf2, NF-κB, Wnt, AP-1, bestrophin-3). Other cellular proteins and processes targeted by ROS/Ca(2+) (metallothioneins, Bcl-2 proteins, ubiquitin-proteasome system, ER stress-associated unfolded protein response, autophagy, cell cycle) can evoke death or survival. Hence, temporary or permanent disruptions of ROS/Ca(2+) induced by Cd(2+) play a crucial role in eliciting, modulating and linking downstream cell death and adaptive and survival signaling cascades.Archives of Toxicology 08/2013; 87(10). · 5.08 Impact Factor
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ABSTRACT: Cd(2+) causes damages to several human tissues. Although the toxicological and carcinogenetic mechanisms of Cd(2+) have been previously established, some basic questions on this toxicant remain unclear. In this study, we constructed Met-cad 1.57, a new fluorescent resonance energy transfer (FRET)-based Cd(2+) indicator, which contains a portion of a Cd(2+)-binding protein (CadR) obtained from Pseudomonas putida as the Cd(2+) sensing key. We produced a human embryonic kidney cell line HEK-MCD157 which stably expresses the Met-cad 1.57 for further investigations. Both fluorescence spectroscopy and FRET microscopic ratio imaging were used to monitor the Cd(2+) concentration within the living HEK-MCD157 cells. The dissociation constant of Met-cad 1.57 was approximately 271 nM. The function of Ca(2+) channels as a potential Cd(2+) entry gateway was further confirmed in the HEK-MCD157 cells. The organelle-targeted property of the protein-based Cd(2+) indicator directly reveals the nucleus accumulation phenomena. In summary, a human kidney cell line that stably expresses the FRET-based Cd(2+) indicator Met-cad 1.57 was constructed for reliable and convenient investigations to determine the Cd(2+) concentration within living cells, including the identification of the entry pathway of Cd(2+) and sub-cellular sequestration.PLoS ONE 06/2013; 8(6):e65853. · 3.53 Impact Factor
Cadmium Induces Transcription Independently of
Intracellular Calcium Mobilization
Brooke E. Tvermoes1,3, Gary S. Bird2, Jonathan H. Freedman1*
1Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health (NIH), Research Triangle Park, North
Carolina, United States of America, 2Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health (NIH), Research
Triangle Park, North Carolina, United States of America, 3Nicholas School of the Environment, Duke University, Durham, North Carolina, United States of America
Background: Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular
mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates
transcription by altering intracellular calcium concentration ([Ca2+]i) and disrupting calcium-mediated intracellular signaling
processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA
chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal.
Methodology/Principal Finding: In the present report, the effects of cadmium on [Ca2+]iunder non-cytotoxic and cytotoxic
conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in
HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium.
Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-
responsive genes but did not affect [Ca2+]imobilization or increase steady-state mRNA levels of calcium-responsive genes.
In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered
calcium-responsive gene expression.
Conclusions/Significance: These data indicate that at low levels, cadmium induces transcription independently of
intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-
responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism.
Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription.
Citation: Tvermoes BE, Bird GS, Freedman JH (2011) Cadmium Induces Transcription Independently of Intracellular Calcium Mobilization. PLoS ONE 6(6): e20542.
Editor: Nai Sum Wong, University of Hong Kong, Hong Kong
Received December 20, 2010; Accepted May 5, 2011; Published June 9, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported in part by the Intramural Research Program of the NIH, and NIEHS (Z01ES102045). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The transition metal cadmium is a persistent environmental
toxicant. Diet, occupation, and smoking are the primary routes of
cadmium exposure to the public. Exposure to this metal is
associated with numerous human pathologies including kidney
dysfunction, osteoporosis, respiratory ailments, and birth defects
[1,2,3,4]. In addition, cadmium is classified as a Type I human
carcinogen, based on animal studies and data indicating that
occupational exposure leads to an increased risk of lung cancer .
The prevalence of cadmium-associated diseases is increasing and
cadmium-induced pathologies are appearing at levels below
current OSHA standards [6,7,8].
In vivo and in vitro exposure to low concentrations of cadmium
(1–5 mM) initiates an adaptive response that ameliorates the metal-
induced toxicity. Toxic effects are reduced by increasing the levels
of multiple stress-response proteins [9,10,11]. Analysis of tran-
scriptome data from multiple species indicates that cadmium
exposure alters the expression of hundreds of genes [9,12,13,14].
Bioinformatic analyses of cadmium-transcriptomes identify the
expected metal-responsive and stress-response processes/pathways
including p38, extracellular signal-regulated kinase (ERK), and
Jun N-terminal kinase (JNK)/mitogen-activated protein kinase
(MAPK) pathways. Other pathways have been identified however,
that cannot be directly associated with metal detoxification or the
repair of metal-induced damage. In addition, the transcription of
hundreds of additional genes is affected at higher, cytotoxic
cadmium concentrations. An analogous process is observed in
HepG2 cells treated with physiological and toxicological concen-
trations of copper .
The ability of cadmium to affect the expression of hundreds of
functionally unrelated genes can be attributed to its capacity to
modulate the activity of multiple signal transduction pathways.
Cadmium activates p38, ERK, and JNK/MAPK pathways .
Activation of MAPK pathways affects the transcription of genes
involved in the stress-response, as well as growth and development.
In addition to the MAPK pathway, cadmium influences the
activities of p53, NRF2, protein Kinase C, casein kinase 2, and
calcium/calmodulin-dependent kinase II (CaMK II) [17,18,
19,20]. Cadmium may also influence gene expression by affecting
the levels of second messengers, such as reactive oxygen species,
cAMP and calcium.
PLoS ONE | www.plosone.org1 June 2011 | Volume 6 | Issue 6 | e20542
It has been suggested that cadmium-activation of ERK, p38,
and JNK results in part from an elevation of intracellular calcium
concentration ([Ca2+]i) [21,22]. While several studies indicate that
exposure to cadmium causes increased [Ca2+]i, the mechanism by
which cadmium affects [Ca2+]iremains poorly understood .
Several factors have made defining the precise effects of cadmium
on [Ca2+]iproblematic. A major issue has been the use of the
calcium chelator 1, 2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tet-
raacetic acid (BAPTA) and BAPTA-based fluorescent calcium
indicators. The BAPTA-based indicators and chelators are able to
bind cadmium with high affinity. BAPTA binds calcium with a Kd
,0.2 mM, however it also binds cadmium, but with Kd,1 pM. In
addition, the fluorescent intensity of cadmium-bound Fura-2, a
common BAPTA-based fluorescent dye used to monitor [Ca2+]i, is
70% greater for the cadmium-bound form compared to the
calcium-bound form . A second confounding factor is the use
of cytotoxic concentrations of cadmium. LD50s for cadmium in
mammalian cells are ,10 mM, while studies examining the effects
of cadmium on [Ca2+]iroutinely expose cells to concentrations of
metal in far excess of this level [18,21,24]. Thus, there is a need to
better understand the relationship between cadmium exposure,
calcium mobilization, and the subsequent effect on transcription.
In the current report, the effect of cadmium on [Ca2+]i is
examined using the protein-based calcium sensor, yellow came-
leon (YC)3.60, which is constitutively expressed in HEK293 cells
(HEK293::YC3.60) . The yellow cameleon does not respond
to changes in intracellular cadmium concentration ([Cd2+]i).
Exposing HEK293 cells to 1 mM cadmium for 4 h was sufficient
to induce transcription of several cadmium-responsive genes, but
did not affect cell viability, intracellular calcium levels, or the
transcriptional activity of calcium-responsive genes. In contrast,
exposure to cytotoxic levels of cadmium, 30 mM for 4 h,
significantly decreased cell viability, reduced endoplasmic reticu-
lum (ER) calcium stores, and significantly altered the transcrip-
tional activity of calcium-responsive genes. These data indicate
that non-cytotoxic concentrations of cadmium induce transcrip-
tion independently of intracellular calcium mobilization. Further-
more, only when cells are exposed to cytotoxic cadmium
concentrations is calcium mobilized from ER pools.
Cadmium-inducible transcription in HEK293::YC3.60 cells
qRT-PCR of three well-characterized cadmium-inducible
genes: mt-1 (metallothionein-1), c-fos, and grp-78 (78-kDa glucose-
regulated protein/HSPA5) was used to quantify the transcriptional
response of HEK293::YC3.60 cells exposed to cadmium or with
altered [Ca2+]i. Exposure of HEK293::YC3.60 cells to 1 or 30 mM
cadmium resulted in a rapid and significant increase in mt-1
mRNA levels (Fig. 1). This observation was consistent with
previous studies where cadmium exposure produced an increase in
the steady-state levels of mt-1 mRNA [11,18]. Treatment with
thapsigargin also caused an increase in mt-1 mRNA, but only
following a 24 h exposure. Thapsigargin is a potent inhibitor of
calcium ATPases of the endoplasmic reticulum (ER), leading to a
depletion of ER calcium and a concurrent increase in [Ca2+]i
c-fos mRNA levels significantly increased following 1 h cadmi-
um and thapsigargin exposures (Fig. 1). Longer exposures to 1 mM
cadmium resulted in a gradual decrease in the steady-state level of
c-fos mRNA, where at 24 h the mRNA level was not significantly
different from control cells. The elevated levels of c-fos mRNA
following 24 h exposures to 30 mM cadmium and thapsigargin
may be the result of non-specific stress-responses (see below).
The steady-state level of grp-78 mRNA increased in response to 1
and 30 mM cadmium and thapsigargin(Fig. 1). Thapsigargin had the
fastest and largest effect on grp-78 mRNA levels, followed by 30 mM
cadmium. Similar to c-fos, exposure to 1 mM cadmium caused a
transient elevation in grp-78 which returned to baseline by 24 h.
Figure 1. Effects of cadmium and thapsigargin on transcrip-
tion. Total RNA was isolated from HEK293::YC3.60 cells exposed to
either 1 mM (square) or 30 mM (triangle) cadmium, or 2 mM thapsigargin
(circle) for 1, 4, or 24 h. Steady-state mRNA levels of mt-1, c-fos, and grp-
78 were measured using qRT-PCR. All measurements were normalized
to mRNA levels of actin. Fold change was normalized to mRNA levels
observed in control cells. Results were mean log2fold change 6 SEM
(n=3) and were analyzed by one-way ANOVA followed by Dunnett’s
post-test; single (*) and double (**) asterisks indicate significant
differences from controls at p,0.05 and p,0.001, respectively.
Effect of Cadmium on Intracellular Calcium
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The effect of cadmium and altered [Ca2+]ifollowing thapsi-
gargin on the steady-state levels of mt-1, c-fos, and grp-78 mRNAs
in HEK293::YC3.60 cells were similar to those reported in
previous studies . These data indicated that this cell line would
be an appropriate system to investigate mechanisms of cadmium-
induced transcription and a potential contributory role of calcium.
Intracellular cadmium accumulation and cell viability
Cellular uptake of cadmium in HEK293::YC3.60 cells was
monitored using fura-5F, which binds cadmium with high affinity
. Prior to cadmium exposure, intracellular calcium stores were
depleted by incubating cells for 5 min with 10 mM ionomycin, in
calcium-free HBSS. This would ensure that changes in fluores-
cence ratios represented cadmium influx rather than changes in
[Ca2+]i. The addition of 2 mM cadmium resulted in a rapid and
significant increase in the fluorescence ratio (Fig. 2A). This
indicated that cadmium entered the cells and that fura-5F was
capable of detecting changes in [Cd2+]i.
To assess intracellular cadmium concentration ([Cd2+]i) levels in
chronically treated cells, cells were exposed to various concentra-
tions of cadmium for either 4 or 24 h and then loaded with fura-
5F (Fig. 2B). Cadmium accumulation was both time- and
concentration-dependent. The fura-5F fluorescence ratio showed
the largest increase in cells exposed to 30 mM cadmium for 24 h.
Alterations in [Ca2+]ioccur during cadmium-induced cell death
[21,24,29,30,31,32,33]. Therefore, the effect of cadmium on
HEK293::YC3.60 cell viability was assessed (Fig. 2C). Exposure to
1 or 3 mM cadmium for 4 h or 24 h did not significantly affect the
number of viable cells. These conditions were defined as non-
cytotoxic cadmium concentrations. In contrast, exposure to higher
cadmium concentrations for either 4 or 24 h significantly reduced
cell viability compared to control cells and were therefore defined
as cytotoxic cadmium concentrations. This suggests that changes
in [Ca2+]iat 10 or 30 mM cadmium may be a consequence of on-
going cell death and not a direct activity of cadmium to induce
intracellular calcium release.
Effect of cadmium on YC3.60 expressed in HEK293 cells
The ability of YC3.60, a protein-based calcium ion sensor, to
detect changes in [Cd2+]iwas assessed in HEK293::YC3.60 cells.
Figure 3 presents a time course of a typical biphasic calcium
response in HEK293 cells expressing YC3.60 in calcium-free
media. Similar to studies using fura-5F loaded HEK293::YC3.60
cells (Fig. 2A), the addition of ionomycin produced a transient
increase in [Ca2+]i. In addition, supplementing the medium with
external calcium produced an influx of calcium, as indicated by a
rapid and sustained increase in the fluorescence ratio (Fig. 3). In
contrast to studies using fura-5F-loaded cells, the addition of
2 mM cadmium following ionomycin treatment did not affect the
fluorescence ratio. These results demonstrated that YC3.60 could
distinguish between calcium and cadmium ions; thus it would be a
useful tool to measure the effects of cadmium on [Ca2+]i.
Effect of cadmium on ER calcium stores and calcium
It has been proposed that cadmium activates transcription by
depleting ER calcium stores to alter [Ca2+]i. To determine if
non-cytotoxic concentrations of cadmium had an effect on calcium
homeostasis or ER calcium stores, HEK293::YC3.60 cells were
treated with the SERCA pump inhibitor thapsigargin prior to
cadmium addition. Treating HEK293::YC3.60 cells with thapsi-
gargin resulted in a typical biphasic change in [Ca2+]i, which
represented depletion of ER calcium stores and the re-entry of
calcium across the plasma membrane by the activation of store-
operated calcium entry (SOCE) (Fig. 4A) [35,36]. The biphasic
calcium response induced by thapsigargin allowed several aspects
of calcium homeostasis to be investigated: (i) thapsigargin-
mediated inhibition of SERCA pump activity; (ii) proper operation
of plasma membrane calcium-ATPases; and (iii) the activation
mechanism and permeation properties of SOCE . To
determine if cadmium exposure altered the activity of these
homeostatic processes, the thapsigargin-induced biphasic calcium
response in cells exposed to 1 mM cadmium for 4 h was compared
to control cells. The biphasic calcium response in HE-
K293::YC3.60 cells exposed to 1 mM cadmium was not
significantly different from control cells (Fig. 4B). This indicated
that exposure to cadmium at a concentration sufficient to induce
transcription did not disrupt calcium homeostasis.
Any decrease in ER calcium store content would likely be
reflected in a diminished thapsigargin-induced [Ca2+]i peak.
Exposure to cadmium however, did not significantly affect the
thapsigargin-induced peak, which suggested that cadmium did not
deplete ER calcium stores (Fig. 4B).
To further investigate the effect of cadmium on ER calcium
stores, an ionomycin depletion protocol was utilized . In the
absence of extracellular calcium, ionomycin (10 mM) selectively
triggers a rapid release of ER calcium stores . The height of
the ionomycin-induced [Ca2+]ipeak is approximately proportional
to the ER calcium content (Fig. 4C). By comparing peak heights,
the effects of various concentrations and exposure times of
cadmium on ER calcium stores were determined. Following 4 or
24 h exposures, concentrations below 30 mM cadmium did not
significantly affect ER calcium stores. A decrease of ER calcium
store content was observed, however, in cells treated with 30 mM
cadmium (Fig. 4D). The levels of ER calcium stores were reduced
by 36% and 57% following 4 and 24 h exposures, respectively.
These data suggest that low concentrations of cadmium
(1–10 mM), which induced changes in gene transcription (Fig. 1),
did not interfere with calcium signaling mechanisms nor deplete
ER calcium stores.
Effects of cadmium and thapsigargin on cAMP/Ca2+
responsive gene expression
PCR arrays were used to assess the ability of cadmium to affect
the steady-state levels of mRNAs encoded by 84 cAMP/calcium -
responsive target genes. Consistent with the [Ca2+]imeasurements
(Fig. 4), 1 mM cadmium did not significantly affect the transcrip-
tion of the majority of the cAMP/calcium-responsive genes
(Tables 1 and 2). Exposure to 30 mM cadmium for 4 or 24 h,
however, significantly affected the mRNA levels of many of the
cAMP/calcium -responsive target genes. This response may be the
result of 30 mM cadmium depleting ER calcium stores thus
increasing [Ca2+]i (Fig. 4D), or a consequence of cadmium-
mediated cell death (Fig. 2C), which also alters [Ca2+]i. To
determine whether the ability of cadmium to induce transcription
was due to changes in [Ca2+]i, the patterns of cadmium-responsive
gene expression were compared to those produced following
thapsigargin exposure (Table 3). Exposure to 1 mM cadmium
resulted in the differential expression of four genes that were also
affected by thapsigargin exposure (Fig. 5). For these genes (TNF,
FOS, PTGS2, and ERG1) however, cadmium exposure caused a
significant decrease in the steady-state level of mRNA, whereas
thapsigargin exposure produced a significant increase in mRNA
For 30 mM cadmium, the expression of eleven genes was
significantly affected among the three exposure conditions: 4 and
24 h 30 mM cadmium and 4 h 2 mM thapsigargin (Fig. 5, Table 4).
Effect of Cadmium on Intracellular Calcium
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Gene Ontology analysis showed that these genes were involved in
apoptosis, differentiation, and mitogenesis. Fifteen of the seventeen
genes induced by thapsigargin were up-regulated in response
to a 24 h exposure to 30 mM cadmium. The majority of the genes
affected by cadmium, however, were not differentially expressed
following thapsigargin exposure. In addition, the expression of
TNF and PER1 was suppressed by cadmium exposure, but
increased following thapsigargin exposure. Together, these results
suggested that exposure to cytotoxic concentrations of cadmium
affected transcription via a mechanism that may be independent of
changes in [Ca2+]i.
Cadmium is a well-known toxicant that is continuously
introduced into the environment. Environmental exposure to
cadmium is a substantial public health concern. Recent studies
suggest that incidences of cadmium-associated disease are
escalating in populations exposed to low levels of cadmium
[6,8,40,41]. To more fully understand the relationship between
cadmium and disease, it is imperative to understand the
mechanism of cadmium-responsive transcription, under both
adaptive and toxicological conditions. Toxicological effects at
low-levels of exposure are prevented or repaired by altered
expression of multiple stress-response proteins and their cognate
genes. Alterations in gene expression have been observed in
multiple systems at cadmium concentrations below those leading
to measurable toxicological responses. In vitro exposure of HeLa or
CCRF-CEM cells to non-toxic concentrations of cadmium affects
the expression of ,60 and 20 genes, respectively [42,43].
Treatment of mice with non-toxic doses of cadmium causes the
differential expression of 22 genes . Likewise in C. elegans,
,100 genes are differentially expressed following cadmium
Figure 3. Effect of cadmium on YC3.60 fluorescence in
HEK293::YC3.60 cells. Traces represent changes in YC3.60 fluores-
cence ratios following ionomycin exposure in calcium-free HBSS. At
,15 min, the medium was supplemented with either 2 mM calcium
representative of typical responses observed in four independent
O, black line) or 2 mM cadmium (Cdz2
O, red line). Traces are
Figure 2. Cadmium uptake and cell viability in HEK 293::YC3.60
cells. A, Fura-5F loaded cells were incubated with ionomycin for 10 min
to deplete intracellular calcium stores and then 2 mM cadmium in
calcium-free HBSS was added to the medium (solid line). In the
experiment represented by the dashed line, similar conditions were
used except cells were not exposed to ionomycin prior to cadmium
addition. Traces are representative of typical responses observed in at
least three independent experiments. B, HEK293::YC3.60 cells were
exposed to 0, 1, 3, 10, and 30 mM cadmium for 4 (closed circle) and 24
(open circle) h. Following metal exposure, cells were incubated with
fura-5F and then fluorescence ratios were determined. Asterisks
indicate a significant (p,0.001) difference between control and
cadmium exposed groups. Data were analyzed by two-way ANOVA
followed by Tukey’s post-test. Asterisks indicate a significant (p,0.001)
difference between control and cadmium exposed groups. C, Cell
viability of HEK293::YC3.60 cells exposed to 0, 1, 3, 10, and 30 mM
cadmium for 4 (closed circle) and 24 (open circle) h. Data were expressed
as the mean 6 SEM and were analyzed by one-way ANOVA followed by
Dunnett’s post-test. Asterisks indicate significant (p,0.001) difference
from control and cadmium exposed groups.
Effect of Cadmium on Intracellular Calcium
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exposure under conditions that do not induce a general stress
At toxic concentrations of cadmium, the transcription of
hundreds of genes is affected. Among the hundreds of cadmium-
responsive genes, many of the cognate regulatory pathways have
been identified. These pathways include MAPK, p53, NRF2,
Protein Kinase C, casein kinase 2, and CaMK II [17,18,19,20].
Although regulatory pathways have been identified, the molec-
ular mechanisms by which cadmium initially activates these
pathways to elicit specific transcriptional changes have not been
One hypothesis that addresses how cadmium activates intra-
cellular signaling pathways proposes that the metal modulates the
level of [Ca2+]i. Thus, calcium could be viewed as a second
messenger that mediates cadmium-responsive transcription. While
several mechanisms have been proposed by which cadmium may
alter [Ca2+]i, the effects of cadmium on calcium signaling remain
ambiguous. This ambiguity may be a consequence of technical
approaches traditionally used to investigate calcium-mediated
signaling processes. Specifically, there are potential problems in
the interpretation of data from studies in which BAPTA or
BAPTA-based fluorescent calcium indicators are used when
examining the consequences of cadmium exposure on [Ca2+]i.
Cadmium binds to these compounds with a .1000-fold higher
affinity and can produce higher fluorescence than calcium making
the interpretation of this data problematic . A loss of a
response during co-exposures of BAPTA with cadmium could be
due to decreases in the effective cadmium concentrations, rather
than effects on [Ca2+]i. Likewise, an increase in fluorescence in
fura-loaded cells following exposure to cadmium could be due to
the binding of cadmium to the dye, rather than a release of
intracellular calcium from storage. To circumvent this problem, a
protein-based calcium indicator, yellow cameleon 3.60 was used in
the current studies. In HEK293::YC3.60 cells, cadmium exposure
did not elicit a change in YC3.60 fluorescence (Fig. 3). Under
similar experimental conditions however, cadmium produced
significant increases in fura-5F fluorescence ratios (Fig. 2). This
indicates that fura-5F and potentially other BAPTA-based
Figure 4. panel A, Traces represent [Ca2+]imeasured in control HEK293::YC3.60 cells (black line) or cells following exposure to 1 m mM
cadmium for 4 hr (red line). The traces are representative of typical responses observed in at least three independent experiments. panel B, Means
of the peak values in thapsigargin response following exposure to 1 mM cadmium for 4 hr (gray bar) or non-cadmium treated (black bar). Data were
expressed as the mean 6 SEM and were analyzed by an unpaired t-test. There were no significant differences between control and the cadmium
exposed groups. panel C, Traces represent [Ca2+]imeasured in HEK 293::YC3.60 cells under control conditions (black line) or following a 4 h exposure
to 1 mM cadmium (red line). Following incubation with cadmium, cells were treated with an ionomycin-BAPTA solution in calcium-free HBSS. The
traces were representative of typical responses observed in at least three independent experiments. panel D, Mean peak values of ionomycin
responses in HEK293::YC3.60 cells following exposure to 0, 1, 3, 10, and 30 mM cadmium for 4 h (black bar) or 24 h (gray bar). Data were expressed as
the mean 6 SEM and were analyzed by one-way ANOVA followed by Dunnett’s post-test. Asterisks (*) indicate significant difference (p,0.001)
between control and cadmium exposed groups.
Effect of Cadmium on Intracellular Calcium
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fluorescent dyes can be used to measure [Cd2+]i, but are not
appropriate when measuring the effects of cadmium on [Ca2+]i.
To assess the effects of cadmium on intracellular calcium
homeostasis, HEK293 cells that stably expressed YC3.60 were
used. YC3.60 provides a direct measure of [Ca2+]i without
interference from cadmium (Fig. 3). A second consideration in the
current experimental design is the use of non-cytotoxic concen-
trations of metal. Exposing HEK293::YC3.60 cells to 1 mM
cadmium for 4 h was sufficient to increase steady-state mRNA
levels of three well-characterized cadmium-responsive genes
(Fig. 1). In addition, exposure to 1 mM cadmium for 4 or 24 h
did not produce any significant toxicological responses (Fig. 2C).
Based on these results, subsequent calcium homeostasis and
signaling experiments were performed using HEK293::YC3.60
cells exposed to 1 mM cadmium. To replicate previous studies and
gain an understanding of how cytotoxic levels of cadmium affect
[Ca2+]i, cells were also exposed for 4 and 24 h to 30 mM
cadmium, which is approximately three-times the 24 h LC50for
this cell line.
Using this experimental design, low-dose cadmium exposures
did not interfere with calcium homeostasis nor deplete ER calcium
store content (Fig. 4). Only high concentrations of cadmium
(30 mM) depleted ER calcium stores. This is similar to that
reported by Biagioli et al., who observed a significant depletion of
ER calcium stores in NIH 3T3 cells treated with 15 mM cadmium
for 12 h . The reported LC50s for cadmium in 3T3 cells range
from 1–5 mM . These results suggest that as cells succumb to
metal toxicity, calcium is released from intracellular stores.
Cadmium exposure increases the activity of MAPK and CaMK
II regulated pathways [16,17,20,21,46]. Since MAPKs and CaMK
II are considered integrators of calcium signaling, the effect of
cadmium on the expression of calcium responsive genes was
investigated using cAMP/calcium signaling focused arrays.
Exposure of HEK293::YC3.60 cells to non-cytotoxic levels of
cadmium, 1 mM for 4 or 24 h, did not affect the expression of a
significant number of genes. One gene was commonly affected by
both non-cytotoxic cadmium conditions and thapsigargin-induced
intracellular calcium release. Following 24 h exposure to1 mM
cadmium, an additional three genes were affected by both
cadmium and thapsigargin. However, among the commonly
affected genes; TNF, FOS and EGR1; cadmium caused a
significant decrease in their steady-state mRNA levels while an
increase in [Ca2+]ihad the opposite effect (Table 2). These results
are consistent with the lack of a significant effect on [Ca2+]iin
cells exposed to low concentrations of cadmium (Fig. 4). These
metal concentrations are associated with adaptive responses and
do not deplete ER calcium stores nor interfere with intracellular
calcium signaling. Thus under these conditions calcium does not
function as a second messenger mediating cadmium-responsive
Exposure to cytotoxic levels of cadmium affected the steady-
state mRNA levels of ,60% of the genes, in contrast to non-
cytotoxic conditions that affected ,2% (Fig. 5, Table 1 and 2).
These results are similar to previous studies demonstrating
concentration-dependent increases in the number of affected
genes when cells are exposed to environmental toxicants; i.e., as
the concentration of toxicant increases from adaptive to cytotoxic,
there is a concomitant increase in the number of genes whose
steady-state level of expression change. This was observed in
HepG2 cells exposed to copper; where at physiological copper
concentrations (200 mM) the expression of 30 genes was affected,
but at toxicological concentrations (600 mM) the number of
affected genes increased to 790 .
Exposure to 30 mM cadmium for 24 h affected the expression of
50 genes. This result was consistent with the [Ca2+]imeasurements
in which 30 mM cadmium affected ER calcium stores (Fig. 4). The
majority of the thapsigargin-inducible genes were also affected by
30 mM cadmium. However, three-times as many genes were
affected by cadmium as thapsigargin, 17 vs. 53 (Fig. 5). In
addition, the steady-state mRNA levels of TNF and PER1
increased in response to intracellular calcium release, but
decreased following cadmium exposure. This suggests that the
overlap among affected genes may be the result of a general
activation of transcription by cadmium rather than a specific
Exposure to 30 mM cadmium caused a significant decrease in
cell viability and depletion of ER calcium stores. At cytotoxic
concentrations, calcium release may not be a specific cadmium-
induced response; rather it could be a secondary or tertiary
response, or non-specific affect. For example, cadmium exposure
in rodents causes an increase in cAMP levels by increasing
adenylate cyclase and decreasing cAMP phosphodiesterase
activities, which ultimately leads to the activation of cAMP-
dependent protein kinase regulated genes . Similarly, the
activation of DNA damage response is due to cadmium-induced
DNA damage via oxidative stress and inhibition of DNA repair,
Table 1. SuperArray analysis of cAMP/calcium signaling in
cells exposed to cadmium for 4 h.
Gene Name Fold Changep value
1 m mM Cadmium (4 h)
30 m mM Cadmium (4 h)
PENK 4.664 0.000512
ATF3 4.6518 0.000101
FOSB 3.0299 0.001182
GEM 2.7639 0.000113
PER1 -2.0993 0.00208
Effect of Cadmium on Intracellular Calcium
PLoS ONE | www.plosone.org6June 2011 | Volume 6 | Issue 6 | e20542
and not a direct interaction between cadmium and p53 . In
addition, the activation/suppression of transcription could be a
consequence of metal-induced membrane damage and cell death.
As a consequence of the breakdown of intracellular structures,
calcium would be released from membrane-bound intracellular
stores. Cadmium-induced oxidative stress and lipid peroxidation
occur within minutes of exposure to toxic concentrations of metal
and prior to any measurable cytotoxicity [49,50,51]. Metal-
induced damage could activate multiple processes. The activation
of signaling proteins and cognate regulatory pathways would affect
the expression of dozens of genes including the calcium/cAMP
responsive genes on the array.
Low-level exposure to cadmium is relevant to human health as the
general population is constantly exposed to low levels of this metal.
Exposure to non-cytotoxic levels of cadmium is sufficient to affect
gene expression, but does not alter calcium homeostasis. In addition,
the transcription of calcium/cAMP responsive genes is unaffected by
non-cytotoxic levels of cadmium. These data strongly suggest that
Table 2. SuperArray analysis of cAMP/calcium signaling in
cells exposed to cadmium for 24 h.
Gene Name Fold Changep value
1 m mM Cadmium (24 h)
30 m mM Cadmium (24 h)
POU1F1 20.8117 0.000247
PTGS2 20.6576 0.000475
GIPR 12.922 0.00001
THBS1 8.3133 0.000252
DUSP1 4.5155 0.000003
Table 2. Cont.
Gene NameFold Changep value
CREB1 -5.9272 0.052032
Table 3. SuperArray analysis of cAMP/calcium signaling in
cells exposed to thapsigargin.
2 m mM Thapsigargin (4 h)
Effect of Cadmium on Intracellular Calcium
PLoS ONE | www.plosone.org7June 2011 | Volume 6 | Issue 6 | e20542
cadmium-activated transcription is independent of intracellular
calcium signaling. The results also support the hypothesis that at
cytotoxic concentrations of cadmium, calcium-regulated signaling is
affected as part of a general downstream response to cadmium-
calcium homeostasis. They also suggest that further examination of
the molecular mechanisms regulating cadmium-responsive transcrip-
tion should be conducted at non-cytotoxic metal concentrations,
which are environmentally relevant, and confirm that experimental
reagents do not interact with cadmium.
Materials and Methods
HEK293 cells stably expressing the calcium sensitive protein
yellow cameleon 3.60 (HEK293::YC3.60) were generated by
transfecting HEK293 cells with YC3.60 cDNA using lipofecta-
mine 2000 according to manufacturer’s instructions (Invitrogen/
Life Technologies, Carlsbad, CA). Details about the YC3.60
plasmid and its construction can be found in Nagai, et al. .
Following a 48 h recovery period, transfected cells were sorted on
a FACSVantage SE Flow Cytometer (BD Bioscience, San Jose,
CA) to select and enrich the cell population for cells expressing
YC3.60. The sorting and enriching procedure was repeated three
times to establish a population of cells homogeneously expressing
YC3.60. Subsequently, HEK293::YC3.60 cells were maintained
in culture at 37uC in a 5% CO2 atmosphere in Dulbecco’s
modified Eagle’s medium supplemented with 2 mM glutamine,
10% fetal bovine serum, and 75 mg/ml G-418 (Invitrogen/Life
Technologies). Cells stably expressing YC3.60 were used for all
cell culture experiments.
Cell viability assays
Sensitivity of HEK293::YC3.60 cells to increasing concentra-
tions and exposure times to cadmium was determined by using the
neutral red assay as previously described . HEK293::YC3.60
cells were exposed to 0, 1, 3, 10, or 30 mM cadmium for 4 or 24 h.
Quadruplicate experiments were performed at each concentra-
tion. Results are presented as a percentage of control values,
mean 6 standard mean error (SEM) (n=4). Data were analyzed
by one-way ANOVA followed by Dunnett’s Multiple Comparison
Isolation of total RNA and qRT-PCR
To assess the effects of cadmium and thapsigargin on trans-
cription in HEK293::YC3.60 cells, steady-state levels of mt-1, c-fos,
and grp-78 mRNA were determined using quantitative real time
PCR (qRT-PCR). Cells were treated with cadmium (1 or 30 mM)
or thapsigargin (2 mM) for the last 1, 4, or 24 h of 48 h
incubations. Total RNA was then isolated from both treated and
control cells using RNeasy Mini Kits following manufacturer’s
instructions (Qiagen, Inc., Valencia, CA).
For qRT-PCR, total RNA from three independent experiments
was isolated from treated and control cells. Each biological
replicate was measured in triplicate by two-step qRT-PCR using
SuperScript First-Strand Synthesis System for RT-PCR (Invitro-
gen) and SYBR Green (Qiagen) as previously described . Fold
changes in mRNA levels were calculated using the DDCt method
with b-actin as reference mRNA [54,55]. Changes in gene
expression for cadmium-treated cells were compared to the level of
expression in non-treated cells. Changes in gene expression for
thapsigargin-treated cells were compared to DMSO vehicle-
treated cells. Results are presented as mean log2(fold change) 6
SEM. Sequences for primers used in the qRT-PCR are presented
in Table S1.
Measurements of intracellular calcium concentration
For [Ca2+]imeasurements, log-phase cells were transferred onto
30 mm round glass coverslips and allowed to attach in a small
volume of medium for 4–6 h. Additional DMEM was then added
and the cells incubated for 24–36 h before [Ca2+]imeasurements.
Fura-5F-based calcium measurements.
in the dark with fura-5F by incubating coverslips, mounted in a
Teflon incubation chamber, with 1 mM fura-5F/AM in DMEM at
37uC for 25 min. Immediately before [Ca2+]imeasurements, cells
were washed three times and incubated for 15–30 min at room
temperature in HEPES-buffered salt solution (HBSS; 120 mM
NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 10 mM
glucose in 20 mM HEPES, pH 7.4). Teflon chambers were then
mounted onto a Nikon TS-100 inverted microscope equipped with
Cells were loaded
Figure 5. Venn diagram illustrating genes whose steady-state
levels of expression change in HEK293::YC3.60 cells following
exposure to 1 or 30 m mM cadmium for 4 or 24 h, or 2 mM
thapsigargin for 4 h. The identity and description of the eleven
common genes are presented in Table 4.
Effect of Cadmium on Intracellular Calcium
PLoS ONE | www.plosone.org8June 2011 | Volume 6 | Issue 6 | e20542
a 20X objective (0.75 NA). In experiments where cells were
incubated in nominally calcium-free buffer, HBSS with no added
CaCl2 was used. Fluorescence images were recorded and
analyzed, as previously described . Changes in intracellular
calcium are represented by changes in the ratio of the fura-5F
fluorescence at 340 nm to that at 380 nm (F340/F380). Ratio
changes were obtained from multiple regions of interest (ROI),
where each ROI represented an individual cell. Typically, 25 to 35
ROIs were monitored per experiment. Ratio values were
corrected for autofluorescence, which was determined after
treating cells with 10 mM ionomycin and 20 mM MnCl2.
Because BAPTA-based calcium indicators bind cadmium with
high affinity, fura-5F-loaded HEK293::YC3.60 cells were also
used to monitor the accumulation of [Cd2+]i.
YC3.60-based calcium measurements.
cellswere mountedin Teflon
maintained in HBSS. Chambers were mounted on a Zeiss LSM
510 confocal microscope equipped with a 20X objective (NA 0.8).
The YC3.60 FRET signals were monitored by exciting at 458 nm
and collecting emission images at 490 nm for CFP and 530 nm for
YFP . After correcting for background fluorescence, [Ca2+]i
levels were monitored by calculating the fluorescence ratio of YFP
to CFP emissions (F530/F490). An increase in [Ca2+]iwas observed
as an increase in the F530/F490 ratio. Typically, fluorescence
signals from 25–30 ROIs were monitored for a single experiment.
Effects of cadmium on intracellular calcium pools and
store-operated calcium entry
The status of intracellular calcium pools following exposure to 0,
1, 3, 10 or 30 mM cadmium for 4 or 24 h was assessed by exposing
HEK293 cells to 10 mM ionomycin in the presence of 3 mM
BAPTA . Following incubation in cadmium containing buffer,
cells were mounted in a Teflon incubation chamber and
maintained in HBSS for ,10 min before being treated with
10 mM ionomycin in the presence of 3 mM BAPTA . The
transient calcium response was indicative of the size of intracellular
calcium pool and could be quantified by calculating the peak
YC3.60 response. The peak YC3.60 response was defined by the
maximal response minus the resting fluorescence ratio.
To investigate the effects of cadmium on SOCE, a ‘‘calcium re-
addition’’ protocol was used in HEK293::YC3.60 cells treated
with 2 mM thapsigargin . In cases where the cadmium
concentration was cytotoxic, YC3.60 fluorescence was monitored
only in recognizably viable cells. All data were analyzed by one-
way ANOVA followed by Dunnett’s Multiple Comparison post-
Human cAMP/calcium Signaling RT2ProfilerTMPCR Array
Arrays (SABiosciences, Frederick, MD) were used to examine
the effect of cadmium on calcium-responsive transcription in
HEK293::YC3.60 cells. These arrays contain 84 genes that are
reported to be responsive to changes in cAMP and calcium levels.
The layout and description of the genes on the array are presented
in Table S2.
Cells were grown and RNA was isolated as described above.
The purity and quality of RNA was assessed using the RNA 6000
LabChip and Agilent 2100 Bioanalyzer (Agilent Technologies,
Santa Clare, CA, USA). Procedures for qRT-PCR and array
analysis were performed according to manufacturer’s instructions.
Array data was normalized to the average threshold cycle (Ct)
value of three housekeeping genes: b-2-microglobulin (B2M),
hypoxanthine phosphoribosyltransferase (HPRT1), and ribosomal
protein L13a (RPL13A). These genes were chosen for normaliza-
tion because their Ctvalues did not differ by more than one cycle
among all of the samples and treatments. The average Ctvalue for
the three housekeeping genes did differ by more than one cycle in
cells treated with 30 mM cadmium for 24 h.
To make a meaningful biological analysis of differentially
expressed genes in cells treated with 30 mM cadmium for 24 h, a
normalization factor was calculated for each of the three
independent experiments (0.91, 0.88, and 0.89) so that the
normalized expression ratio of the average Ct value for each
housekeeping gene was equal across compared samples (cadmium
Table 4. Genes affected by both cadmium and thapsigargin exposures.
DDIT3DNA damage-inducible transcript 3 Transcription factor that promotes cell death during ER stress
PTGS2 Prostaglandin-endoperoxide synthase 2Responsible for the prostanoid biosynthesis involved in inflammation and
HSPA5Heat shock 70kDa protein 5 Involved in the folding and assembly of proteins in the ER during stress
TNFTumor necrosis factorMultifunctional proinflammatory cytokine that is mainly secreted by
INHBA Inhibin beta A subunit Member of the transforming growth factor-beta superfamily that may acts as
both a growth/differentiation factor and a hormone
FOSBFBJ murine osteosarcoma viral oncogene homolog B Part of the transcription factor complex AP-1 and regulates cell proliferation,
differentiation, and transformation
PPP1R15AProtein phosphatase 1, regulatory (inhibitor) subunit 15A Helps mediate apoptosis following stressful conditions
GEMGTP binding protein over-expressed in skeletal musclehttp://www.
Belongs to the RAD/GEM family of GTP-binding proteins, could play a role in
receptor-mediated signal transduction
EGR1 Early growth response 1http://www.genenames.org/data/hgnc_data.
Transcriptional regulator of genes required for differentiation and mitogenesis
ATF3Activating transcription factor 3 Member of the CREB protein family and mediates pro-apoptotic effects of p38
FOSFBJ murine osteosarcoma viral oncogene homolog Part of the transcription factor complex AP-1 and regulates cell proliferation,
differentiation, and transformation
aName and description were determined using GeneCards .
Effect of Cadmium on Intracellular Calcium
PLoS ONE | www.plosone.org9 June 2011 | Volume 6 | Issue 6 | e20542
treated vs. control). This normalization factor was then used to
appropriately scale the expression value for each of the 84 calcium
specific genes within each array for cells treated with 30 mM
cadmium for 24 h. These normalized expression values were then
used to determine fold change. Data was then processed with
SABiosciences web-based RT2
Analysis to calculate Ct and relative gene expression values
according to the DD Ct method . A list of differentially
expressed genes was identified using a Student’s t-test. Only two-
fold or greater changes in gene expression with a p,0.05 were
ProfilerTMPCR Array Data
Sequences of primers used for qRT-PCR.
Functional gene grouping of human cAMP/calcium
The authors would like to thank Dr. Atsushi Miyawaki, RIKEN Brain
Institute, Japan, for the generous gift of yellow cameleon 3.60.
Conceived and designed the experiments: BET GSB JHF. Performed the
experiments: BET GSB. Analyzed the data: BET GSB. Contributed
reagents/materials/analysis tools: BET GSB. Wrote the paper: BET GSB
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