Thus, the cadmium-induced sustained activation of ERK could be a response of the cells to survive following cadmium poisoning.. However, treatment of intoxicated cells with U0126, a pote
Trang 1ERK and cell death: cadmium toxicity, sustained ERK
activation and cell death
Patrick Martin and Philippe Pognonec
CNRS FRE3094, Universite´ de Nice Sophia Antipolis, Nice, France
Introduction
Extracellular signal-related kinase (ERK) is among the
most studied proteins, and is involved in many aspects
of cell physiology [1] Discovered 25 years ago [2],
ERK actually consists of two closely related proteins
in vertebrates: ERK1 and ERK2 These proteins are
encoded by two related, but distinct, genes [3,4] ERK
is commonly considered as a key player in mitogenic
signaling following growth factor stimulation [5,6] The
functional specificities of ERK1 and ERK2 have been
investigated for many years, and despite earlier results
suggesting differences, it now appears that both are
functionally indistinguishable [7,8] The originally
reported specificities turned out to be a reflection of
their relative abundances [7] This is also supported by the observation that some species harbor only one ERK gene [7], and by the demonstration that ERK1 and -2 do functionally compensate for one another [9] Nevertheless, thousands of reports (as of today a Medline search with the term ‘ERK [ti]’ produces 3501 articles) have shown that ERK is a central kinase in signal transduction pathways (reviewed in [10]) Its role
in cellular proliferation has been extensively investi-gated, and its ability to protect cells against apoptosis
is also widely accepted The kinetics of ERK activation are biphasic, with a rapid and transient burst of activa-tion (phosphorylaactiva-tion on Tyr and Thr residues) within
Keywords
cadmium; ERK; PKC; sustained activation;
ZIP8
Correspondence
P Pognonec, Transcriptional Regulation and
Differentiation, CNRS FRE3094, Universite´
de Nice, Parc Valrose, 06108 Nice cedex 2,
France
Tel ⁄ Fax: +33 492 07 64 13
E-mail: pognonec@unice.fr
(Received 18 June 2009, revised 30 July
2009, accepted 15 August 2009)
doi:10.1111/j.1742-4658.2009.07369.x
Extracellular signal-related kinase (ERK) is a key player in cell signaling After 25 years of investigation, ERK has been associated with every major aspect of cell physiology Cell proliferation, cell transformation, protection against apoptosis, among others, are influenced by ERK function Surpris-ingly, ERK has also been associated with two apparently opposing pro-cesses The involvement of ERK in cell proliferation has been extensively described, as well as its function in postmitotic cells undergoing differentia-tion The analysis of these apparent discrepancies has led to a more precise understanding of the multiple functions and regulations of ERK More recently, several groups have identified a new and unexpected role for ERK Although being accepted as an important player in the protection against cell death by apoptosis, it is now clear that ERK can also be directly linked to cell death signaling Here, we review the role of ERK in cell response to cadmium and its association with cell toxicity In this sys-tem, ERK is subjected to a continuous activation that can last for days, which ultimately results in cell death Cadmium entry into cells is responsi-ble for this sustained ERK activation, probably via reactive oxygen species production, and protein kinase C has a negative action on this cadmium-dependent ERK activation by modulating cadmium entry into cells
Abbreviations
ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PMA, 4b-phorbol 12-myristate 13-acetate; ROS, reactive oxygen species.
Trang 2minutes of stimulation, followed by a second and more
stable activation that can last for a few hours [11] The
multiple facets of ERK functions have been linked to
the kinetics of activation [12], subcellular localization
[13] and scaffolding proteins [14]
Cadmium poisoning and sustained ERK
activation
Cadmium is a nonessential metal, recognized as a
potent environmental pollutant Its very long
biologi-cal half life (15–30 years) makes it particularly toxic,
as it accumulates in different organs over time,
ulti-mately resulting in diseases such as kidney failure
and⁄ or bone injuries The World Health Organization
[15] defines the critical cadmium concentration in the
kidney cortex at 200 mgÆkg–1 wet cortex, which
approximately corresponds to a 1 mm cadmium
con-centration Above this threshold, kidney dysfunction
appears It was recently discovered that in cells
sub-jected to a low-dose cadmium poisoning, ERK
dis-plays a very unconventional activation pattern In all
cell types tested (primary mouse and rat fibroblasts,
kidney-derived epithelium cell lines, bone
marrow-derived macrophages, primary calvaria cells, HeLa
cells, HEK293 cells, C2C12 cells), a strong ERK
acti-vation was observed 16–24 h after the addition of
micromolar concentrations of cadmium This
activa-tion remained high, for up to 6 days after cadmium
treatment, until the cells died [16] ERK activation has
been associated with protection against apoptosis when
cells are subjected to stressful conditions [17] Thus,
the cadmium-induced sustained activation of ERK
could be a response of the cells to survive following
cadmium poisoning However, treatment of intoxicated
cells with U0126, a potent and specific
mitogen-activated protein kinase⁄ ERK kinase (MEK) inhibitor
that results in the complete inhibition of ERK
activa-tion, following cadmium exposure does not increase
cell death Rather, a low level of cadmium is not as
toxic to cells in the presence of the MEK inhibitor as
it is in its absence In recent years, a similar
observa-tion has been reported by several groups, in
experi-ments performed using relatively low concentrations of
cadmium (generally between 1 and 10 lm) [16,18–21]
This phenomenon is illustrated in Fig 1 and has been
described in detail elsewhere [16] It should be noted
that the decrease in cell number shown in Fig 1
fol-lowing cadmium treatment is the result of cell death
and not due to an inhibition of cell proliferation, as
determined by cell counting as well as quantification of
loss of cell integrity [22] Although the ERK prodeath
effect discussed above has now been reported by
different groups in multiple cell types, it is intriguing
to note that other reports associated ERK with its classical antiapoptotic function following cadmium exposure, particularly in the human nonsmall cell lung carcinoma CL3 cell line [23,24] Intriguingly, others did not see any effect (neither anti- nor proapoptotic)
of ERK activation in response to cadmium, for exam-ple using Clara cells from rat lung [25], mouse brain microvascular endothelial cells (bEnd.3) [26], the human lymphoblastoid Boleth cell line [27] or the human promonocytic U-937 cell line [28] A key point
in explaining these discrepancies could be attributed to the use of different conditions of cadmium treatments Most of the studies reporting a protective role for
A
B
Fig 1 (A) Phenotypic illustration of the implication of ERK activa-tion in cell death following cadmium treatment HEK293 cells were grown for 24 h in control medium (top row) or in the presence of
1 l M CdCl2(bottom row) The cells were also treated with 5 l M
U0126, a potent MEK inhibitor (middle column), 20 l M U0126 (right column) or no inhibitor (left column) This figure illustrates the toxic-ity of cadmium on cells by comparing the two pictures in the left column In addition, the protective effect of blocking ERK activation
is shown in the lower row, where cadmium toxicity decreased in a dose–response manner following U0126 addition The top row indi-cates that U0126 treatment alone had a cytostatic effect on cells,
as fewer cells were found after 24 h treatment with 20 l M U0126 However, please note that with 20 l M U0126, cell density was similar, independent of the presence of cadmium This reflects the protective effect of blocking ERK activation (B) Western blot analy-sis of HEK cell lysates prepared after no treatment (Ø), 24 h 2 l M
CdCl2treatment (Cd), 24 h 10 l M U0126 treatment (U0), 24 h 2 l M
CdCl2 and 10 l M U0126 cotreatment (U0+Cd) The upper panel displays the activated ERK (P-ERK), which was strongly expressed following cadmium treatment, but almost undetectable when cells were cotreated with cadmium and MEK inhibitor U0126 U0126 alone had no effect on ERK activation (U0) The lower panel repre-sents the total amount of ERK proteins, whose level was not affected by the different treatments.
Trang 3ERK used either a higher cadmium concentration
and⁄ or shorter exposure times Further studies will be
required to clarify the reasons for these apparent
dis-crepancies, which could also be cell type related
ERK activation⁄ phosphorylation is the result of the
balance between its phosphorylation by MEK and its
dephosphorylation by dual-specificity phosphatases
[29], among them MKP3 Transiently transfected
tetra-cycline-inducible MKP3 phosphatase in HEK293 cells
results in diminished ERK activation Under these
con-ditions, we found that HEK293 cells were more resistant
to cadmium poisoning as determined by quantifying
the number of adherent cells to those with a rounded
phenotype (P Martin et al., unpublished results)
Taken together, the sustained activation of ERK
can thus be associated with, and is at least partly
responsible for, cell death signaling
Reactive oxygen species (ROS) and ERK
activation
The mechanism responsible for this unusual sustained
activation of ERK remains unclear It is well
estab-lished that cadmium results in a time- and
dose-depen-dent oxidative response of cells by the production of
ROS [30] Several laboratories have found that
pretreat-ment with the N-acetyl cysteine (NAC) antioxidant
effi-ciently protects cells against cadmium toxicity and
abrogates concomitant ERK activation [31,32] This
implication of ROS in ERK activation is strengthened
by a study using differentiated PC12 cells treated with
zinc cations These authors reported a strong activation
of ERK leading to apoptosis that could be alleviated by
antioxidants [33] It has also been demonstrated that
ROS are responsible for Ras activation, which in turn
stimulates its downstream transduction cascade,
includ-ing ERK [34–36] However, the exact mechanisms by
which ROS activate mitogen-activated protein kinase
(MAPK) are still poorly understood Taken together,
these results suggest that ROS production in response
to cadmium does participate in ERK activation
Furthermore, recent data demonstrated that ROS
production also promotes MKP3 degradation via the
ubiquitination⁄ proteasome degradation pathway This
MKP3 degradation correlates with a strong activation
of ERK [37] In addition, phosphatases responsible for
kinase inactivation are also directly inactivated by
oxi-dation of cysteine thiols [38,39] Therefore, the loss of
phosphatase activity is probably at least partly involved
in the sustained activation of ERK following cadmium
poisoning [33,38]
In conclusion, these data strongly suggest that
ROS production following cadmium poisoning results
in both the activation of the MAPK cascade leading
to ERK activation, and in the inactivation of MKP3, which would stabilize ERK activation These two mechanisms could act together to induce the delayed and sustained activation of ERK, which is distinct from the canonical ERK activation observed following growth factor stimulation In this latter case, an immediate response is observed following ligand–receptor interaction, whereas in the case of cadmium intoxication, the progressive accumulation
of ROS would result in the delayed and sustained response of ERK Further studies will be required to validate this model
Cadmium and calcium
In addition to the ROS pathway, other mechanisms are known to be affected by cadmium and result
in ERK activation Long-term exposure to 5 lM cadmium leads to a rapid increase in internal calcium concentration ([Ca2+]cyt) in NIH3T3 cells [40] and in astrocytes This increase in turn leads to an increase
in ROS and to mitochondrial impairment [41] Pretreatment with a calcium chelator reduced ROS production and increased cell survival, indicating that cadmium-induced cell death resulted from disruption
of calcium homeostasis Similarly, a rapid increase in ([Ca2+]cyt) is seen in mesangial cells exposed to cadmium These cells die by autophagy and apoptosis The use of different calcium inhibitors indicated that the release of calcium from the endoplasmic reticulum plays a crucial role in cadmium-induced cell death In addition, the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N¢,N¢ tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) eliminated ERK activation as well as mitochondrial depolari-zation and activation of caspases [42] Together, these results indicate that calcium plays an important role in the cadmium response upstream of ROS production
Sustained ERK activation, apoptosis, autophagy and necrosis
How this sustained ERK activation yields to the onset
of cell death is still controversial, despite indications pointing towards the activation of caspases-3 and -8 [16] It should be noted that although caspase activa-tion has been associated with cadmium poisoning, our recent unpublished results show that blocking caspases with P35, a broad spectrum caspase inhibitor that is very effective in protecting cells from a caspase-8-induced apoptosis [43], does not protect cells from cadmium toxicity, as cell phenotype and cell counts
Trang 4remain indistinguishable from control cells This
obser-vation has also been reported by other laboratories
showing that complete caspase-3 and -8 inhibition
results in blocking poly(ADP-ribose) polymerase
cleav-age, but not reducing cell death [27] Similarly,
treat-ment of PC12 cells with zVAD-fmk, a pan-caspase
inhibitor, had only a limited effect on
cadmium-induced apoptosis [44] Thus, cadmium, in addition to
caspase activation, induces parallel death pathways
that are P35-insensitive and that ultimately result in
cell death High doses (350 lM) of cadmium result in
mitochondrial membrane depolarization within 1 h
and subsequent translocation of apoptosis-inducing
factor into the nucleus [27], which could account for
such a caspase-independent death pathway Kim et al
[21] also reported that inhibition of ERK activation
results in a better survival of murine J774A.1
macro-phages through the strong attenuation of
cadmium-induced necrotic cell death, but not affecting caspase-3
activity and DNA fragmentation Cell death following
cadmium intoxication is therefore not restricted to a
single process, but includes apoptosis, necrosis
and autophagy, depending upon the context of the
intoxication, such as cadmium concentration, as well
as the inducing pathway (lipid peroxidation, ROS
production, internal calcium concentration) [31,42,45]
Although several lines of evidence indicate that
cadmium can result in cell death via multiple
path-ways, the best indication that we have to date to
support the involvement of ERK sustained activation
in cadmium-induced cell death is an engineered
cellu-lar system (Raf-1:ER) in which Raf, an upstream
ERK kinase, is activated at will by the addition of
4-hydroxytamoxifen [46] The induced activation of
Raf in this system results in sustained activations of
MEK and ERK These activations in turn result in
the onset of apoptosis via the death receptor
way, but are independent of the mitochondrial
path-way In this system, P35 protects cells from
apoptosis [46] Together, these results show that
con-stitutive ERK activation per se is sufficient to induce
a cellular response that can ultimately lead to cell
death in certain systems, but that cadmium, in
addi-tion to caspase activaaddi-tion, triggers other parallel
pathways that also culminate in cell death This
could explain why ERK inhibitors, although reported
by several groups as having a survival effect on cells
exposed to cadmium, are usually not sufficient to
completely protect cells In addition, these different
death pathways are probably differentially regulated
depending upon the cell types considered and the
conditions of cadmium exposure [31,32]
Cadmium entry into cells is required for sustained ERK activation and subsequent cell death
Cadmium has long been shown to accumulate in cells However, being a nonessential element, no cadmium-specific transport system exists Cadmium appears to
‘hijack’ some other transport system(s) in cells It is reasonable to propose that other divalent metal cation transporters could be used by cadmium for entry into cells, and consequently, these other divalent cations could compete with cadmium for cell entry and subse-quently protect cells against cadmium poisoning Calcium, copper, magnesium, manganese and zinc divalent metals have been tested as potential competi-tors for cadmium entry Zinc is considered effective in protecting against cadmium poisoning [47] In our hands, although zinc has a significant, but somewhat modest, effect on the different cell types tested, manga-nese is by far the most effective competitor This was demonstrated by intracellular cadmium concentration measurements using 109Cd Adding a 10-fold manga-nese molar excess compared with cadmium to the culture medium resulted in a five-fold decrease in intracellular cadmium concentration [48] Under these conditions, cells are phenotypically protected from cadmium toxicity and ERK is not activated This is a strong indication that cadmium entry into cells is necessary for sustained ERK activation, and that this activation is tightly associated with cell death Surpris-ingly, the status of the Ras–Raf–MEK–ERK cascade following cadmium treatment has not yet been pub-lished Our unpublished data indicate that in HEK293 cells exposed to 2 lM cadmium, c-Raf is maximally phosphorylated after 8 h and then reaches undetect-able levels after 48 h; MEK1⁄ 2 also reach a plateau after 8 h and then the level remains unchanged; ERK phosphorylation increases from 8 to 72 h following cadmium addition (P Martin et al., unpublished results) This suggests that kinases upstream of ERK,
at least up to c-Raf, are also triggered by cadmium
Rapid and transient activation of ERK
by protein kinase C (PKC) prior to cadmium treatment protects cells from cadmium intoxication
Sustained ERK activation following cadmium intoxica-tion is linked to cell death Could other ERK activa-tors also play a role in the onset of cell toxicity following cadmium treatment? Cells have been pre-treated with the phorbol ester 4b-phorbol 12-myristate
Trang 513-acetate (PMA), which activates conventional, as
well as novel, PKCs PKCs play a key role in a vast
array of cellular signaling, including the activation of
ERK [49] PMA mimics classical cell stimulation by
growth factors, and PKC activation results in a strong
and transient activation of ERK [50] Interestingly,
when HEK293 cells are pretreated with PMA, their
susceptibility to cadmium is substantially decreased
The delayed and sustained cadmium-dependent ERK
activation is also strongly reduced under these
condi-tions Prolonged PKC activation by phorbol esters is
known to downregulate PKC for at least 24 h [51]
However, it has been shown that the protective effect
of PMA on cadmium is not due to its downregulation,
but rather to a hit-and-run effect on PKC [22]
Accordingly, when GF 109203X, a broad-spectrum
PKC inhibitor, was applied before or together with
cadmium, ERK activation increased more than
three-fold and cells became more sensitive to cadmium, as
evidenced by a decrease in absolute cell number and a
more pronounced rounded phenotype [22] Although
earlier work has reported that GF 109203X is also a
potent inhibitor of ribosomal S6 kinase (RSK) [52], a
kinase downstream of ERK potentially retro-inhibiting
the MAPK cascade, it is nevertheless reasonable to
assume that this GF 109203X effect, opposed to that
of PMA, indicates that PKC activation is not
responsible for the delayed and sustained activation of ERK following cadmium treatment Rather, PKC appears to play a protective role in cells exposed to cadmium, as its early activation by PMA reduces both delayed ERK activation and cadmium toxicity, whereas blocking its activity has the opposite effect (Fig 2) [22] In short, PKC early activation has a pro-tective effect on cadmium toxicity in HEK293 cells [22,49,53] It is intriguing to note that earlier work on cells from rat pulmonary epithelium indicated that pre-treatment with PMA has no significant effect on apop-tosis after a 12 h exposure to 3 and 10 lM cadmium [25] Surprisingly, similar experiments on rat alveolar epithelial cells showed that exposure to a relatively high level of cadmium (20 lM for 24 h) resulted in PKC activation, and that GF 109203X protected cells from apoptosis [54] However, in this cell system, PKC activity is already high in control conditions, and cadmium does not activate PKC further [25], or only marginally with high cadmium doses [54] In contrast, PKC activity is low in HEK293 cells under normal
Fig 2 PKC activity inhibits ERK activation in response to cadmium.
Exponentially growing HEK cells were used as a control or treated
for 24 h with 2 l M CdCl2 As seen by western blotting analysis on
the upper panel with an anti-ERK-P IgG, this treatment resulted in
the appearance of the activated ⁄ phosphorylated forms of ERK.
Cells were also cotreated with 100 ngÆmL –1 PMA and cadmium
(PMA ⁄ Cd 24h) or pretreated with PMA for 8 h and then treated
with cadmium for 24 h In both cases, ERK activation was
signifi-cantly lower than for cadmium alone Alternatively, cells were
cotreated with 2.5 l M of the PKC inhibitor GF 109203X and
100 ngÆmL–1PMA and cadmium (GFX ⁄ PMA ⁄ Cd 24h) or pretreated
with GF 109203X and PMA for 8 h and then treated with cadmium
for 24 h In both cases, ERK activation was significantly higher than
for cadmium alone The lower panel displays the level of total ERK
proteins, which remained unchanged.
Fig 3 Proposition of a schematic model for the ERK response to cadmium poisoning Cadmium enters cells through the ZIP8 trans-porter, whose activity may be under the negative control of PKC [22] Once in the cells, cadmium accumulation results in a progres-sive increase in [Ca 2+ ]cytand subsequently to production of ROS [41] ROS participate in both the activation of ERK [32] and in the inhibition of dual-specificity phosphatases, including MKP3 [37] This may participate in the constitutive and long-term activation of ERK [22] This activation ultimately results in the activation of casp-ases-3 and -8 and in apoptosis [46] ERK activation has also been involved in necrotic cell death [21] and in autophagy [42] Other parallel pathways are also present, as ERK inhibition only partially protects from cell death In these pathways, PKC has been shown
to play a prodeath role [25,54] The dashed lines represent indirect pathways.
Trang 6growth conditions, and is strongly activated following
a 24 h 2 lm cadmium treatment [22] This could
explain the reasons for these discrepancies
Cadmium transport
The zinc transporter, ZIP8, is the main transporter
hijacked by cadmium to enter cells [55] Several lines of
evidence indicate that PKC activation could inhibit
ZIP8 activity Indeed, PMA treatment of cells exposed
to cadmium, in addition to activation of PKC, results in
a lower intracellular concentration of cadmium and
bet-ter cell survival An increased cell survival in the
pres-ence of cadmium is also obtained by ZIP8 knockdown
with specific small interfering RNAs [22] Finally, three
potential sites that could be phosphorylated by PKC
have been identified on the ZIP8 amino acid sequence
Together, these results are compatible with a model in
which PKC would negatively regulate ZIP8 transporter
activity via direct phosphorylation, and limit cadmium
entry into cells Such a direct regulation of transporter
activities by PKC has already been reported [56,57]
Fur-ther work will be required to validate this hypothesis
Conclusion
Cadmium exposure results in a complex response of
cells It involves internal calcium increases [40],
oxida-tive mechanisms [30], gene reprogramming [58], protein
degradation [59] and kinase activation [60] Although
no definitive picture is currently available for ERK
function in response to cadmium, its sustained
activa-tion has been associated with cadmium-induced cell
death in several systems [16,18–21] Particularly,
phar-macological inhibition of low-dose cadmium-induced
ERK activation diminishes cell death, whereas MKP3
exogenous expression diminishes cell death following
cadmium intoxication (P Martin and P Pognonec,
unpublished data) This sustained ERK activation is
probably the result of ROS-dependent inhibition of
ERK phosphatases, as well as upstream ERK
activa-tion How ERK sustained activation ultimately results
in cell death remains poorly understood Caspase-3
and -8 activation has been demonstrated following
cadmium exposure and sustained ERK activation,
reminiscent of the ERK-dependent caspase-3 and -8
activation reported in a synthetic system in which a
long-term activation of ERK can be induced [46]
Taken together with the other biological systems
pre-sented in this minireview series, cadmium poisoning is
a situation in which ERK activation, in addition to its
more traditionally described functions, appears to act
as a proapoptotic factor Figure 3 is a schematic
repre-sentation of our current understanding of ERK involvement in this process
Acknowledgement
We thank Kim Boulukos for kindly accepting to critically read this manuscript, despite her recent career shift from a full-time scientist to a full-time sculptor
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